USOO6197785B1 (12) United States Patent (10) Patent No.: US 6,197,785 B1 JackSOn et al. (45) Date of Patent: Mar. 6, 2001

(54) ALKOXY-SUBSTITUTED COMPOUNDS, 810 108 3/1959 (GB). METHODS, AND COMPOSITIONS FOR 1474 775 5/1977 (GB). INHIBITING PARPACTIVITY WO95/24379 9/1995 (WO). WO95/29895 11/1995 (WO). (75) Inventors: Paul F. Jackson, Bel Air; Keith M. Maclin, Baltimore; Jie Zhang, Ellicott OTHER PUBLICATIONS City, all of MD (US) Damas, Chemical Abstracts, vol. 74, No. 19, Abstract No. (73) Assignee: Guilford Pharmaceuticals Inc., 110,112y, May 1971.* Baltimore, MD (US) (List continued on next page.) * ) Notice: Subject to anyy disclaimer, the term of this patent is extended or adjusted under 35 Primary Examiner Zinna Northington Davis U.S.C. 154(b) by 0 days. (74) Attorney, Agent, or Firm Nixon & Vanderhye P.C. (21) Appl. No.: 09/145,166 (57) ABSTRACT 1-1. A compound, compositions, methods of use, and processes (22)22) FileFilled: Sep.ep. 1,, 1998 of making formulas I: s s Related U.S. Application Data (63) Continuation-in-part of application No. 09/079,508, filed on May 15, 1998, and application No. 08/922.520, filed on Sep. 3, 1997, now abandoned. (51) Int. Cl." ...... C07D 217/24; A61K 31/47 (52) U.S. Cl...... 514309; 514/233.5; 514/299; 544/128; 546/141; 546/183 or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, (58) Field of Search ...... 514/309, 233.5; prodrug, metabolite, Stereoisomer, or mixtures thereof, 546/141, 183; 544/128 wherein: R", when present, is hydrogen or lower alkyl; (56) References Cited R is lower alkyl, aryl, aralkyl, lower alkanoyl, or U.S. PATENT DOCUMENTS -(CH), (CHOH),(CH), A, wherein n is 1-4, y is 0 or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower Re. 31,617 6/1984 Beverung, Jr. et al...... 514/311 alkanoyl, aryl, aralkyl, -NH, -NH-(lower alkyl), 1,895,105 1/1933 Rath et al...... 514/311 2,467692 4/1949 Petrow ...... 514/311 2,612,5032 - - 12 9/1952f UI yot ------514/310 / I ower alkyl)Ikwl / N 3,300,499 1/1967 Lesher ...... 514/311 -N -N -N O or 3,557,119 1/1971 Humber ...... 514/311 Y(lower alkyl), M / 3,573,304 3/1971 Eberle et al. ... 260/250 3,700,673 10/1972 Watson ...... 514/311 3,900,477 8/1975 Philipp et al...... 514/311 -N 3,950,343 4/1976 Philipp et al.. 3,978,066 8/1976 Philipp et al...... 514/311 3,991,064 11/1976 Brown et al...... 514/311 4,031,097 6/1977 Bach et al...... 514/311 Y represents the atoms necessary to form a fused 5- to 4,472,401 9/1984 Kennewell et al. ... 514/511 6-membered ring that is aromatic or nonaromatic and 4,742,171 5/1988 Martin et al...... 514/311 4,902,695 2/1990 Ornstein ...... 514/307 carbocyclic or heterocyclic, 5,041,653 8/1991 Lee et al...... 564/74 Z is (i) —CHRCHR- where R and R are indepen 5,177,075 1/1993 Suto et al...... 514/248 dently hydrogen, alkyl, aryl or aralkyl, 5,338,851 8/1994 Huff et al...... 546/141 (ii) -RC=CR - where R and Rare independently 5,414,001 5/1995 Ireland et al. . ... 514/311 hydrogen, lower alkyl, aryl, aralkyl, chlorine, bro

5,420,136 5/1995 Lewis et al...... 514/311 mine or -NR7R, where R7 and R are indepen 5,618,8135,589.483 12/19964/1997 WestChu et ...... al...... 514/310514/311 dentlvently hwdhydrogen or lower alkyl,Ikvl or, RandR,R and R. taktaken 5,756,510 5/1998 Griffin et al...... 514/311 together, form a fused 5- to 6-membered ring that is 5,767,135 6/1998 Fernandez-Pol ...... 514/354 title or nonaromatic and carbocyclic or hetero FOREIGN PATENT DOCUMENTS (iii,CyCIIC, Ric-N- 628 255 5/1963 (BE). (iv) - CR(O)NR'— O 601 246 6/1978 (CH). (v) -C(O)NR'-. 963 184 5/1957 (DE). O 355 750 2/1990 (EP). O 539 805 5/1993 (EP). 2305 182 10/1976 (FR). 23 Claims, 2 Drawing Sheets US 6,197,785 B1 Page 2

OTHER PUBLICATIONS Zhang et al. Science 263:687-89 1994. A. Albert & J.N. Phillips, “Ionization Constants of Hetero Huang et al. Science 265:1883–1885 1994. cyclic Substances. Part II. Hydroxy-derivatives of Nitrog Banasik et al. Molec. Cell Biochem. 138:185-97 1994. enous Six-membered Ring-compounds”, Journal of the Wallis et al. NeuroReport 5:3, 245-48 1993. Chemical Society, pp. 1294–1304 (1956). Dawson et al. J. Neurosci 13:6, 2651–61 1993. M. Banasik et al., “Specific Inhibitors of Poly(ADP-Ribose) Beckman et al. Biochemical Society Transactions Synthetase and Mono (ADP-Ribosyl) transferase”, Journal 21:330-334 1993. of Biological Chemistry, 267 (3):1569–75 (Jan. 25, 1992). Dawson et al. Proc. Natl. Acad Sci. USA 88: 6368–711991. Chemical Abstracts, vol. 52, No. 17, abstract No. 14606h ( Milam et al. Science 223:589-91. 1984. Sep. 10, 1958). Bauer et al., “Modification of Growth Related Enzymatic Chemical Abstracts, vol. 55, No. 6, abstract No. 5491ce Pathways and Apparent LOSS of Tumorigenicity of a ras (Mar. 20, 1961). transformed Bovine Endothelial Cell Line by Treatment Chemical Abstracts, vol. 58, No. 4, abstract No. 3425d (Feb. with 5-Iodo-6-amino-1,2-benzopyrone (INHBP)”, Intl.J. 18, 1963). Oncol., 8:239–52 (1996). R. Griffin et al., “Novel Potent Inhibitors of the DNA Repair Cosi et al., “Poly (ADP-Ribose) Polymerase (PARP) Revis Enzyme Poly(ADP-Ribose) Polymerase (PARP)", Anti ited. A New Role for an Old Enzyme: PARP Involvement in Cancer Drug Design, 10 (6):507-14 (Sep. 1995). Neurodegeneration and PARP Inhibitors as Possible Neuro A. Nuvole & G. Pinna, “Synthesis of protective Agents”, Ann. N. Y. Acad. Sci.825:366-79 (1997). 1-Amino-4-chloroisoquinoline. A New Approach to 1-Ami Cosi et al., “Poly (ADP-Ribose) Polymerase Inhibitors noisoquinoline”,Journal of Heterocyclic Chemistry, vol. 15, Protect Against MPTP-induced Depletions of Striatal pp. 1513–1514 (Dec. 1978). Dopamine and Cortical Noradrenaline in C57B1/6 Mice', M.J. Suto et al., “Dihydroisoquinolinones: The Design and Brain Res.729:264–69 (1996). Sythesis of a New Series of Potent Inhibitors of Poly(AD Cristovao et al., “Effect of a Poly (ADP-Ribose) Polymerase P-Ribose) Polymerase”, Anti-Cancer Drug Design, vol. 7, Inhibitor on DNA Breakage and Cytotoxicity Induced by pp. 107–117 (1991). Hydrogen Peroxide and Y-Radiation”, Terato, Carcino, E. Taylor et al., “Nucleophilic Displacement of Primary and Muta., 16:219–27 (1996). Amino Groups via 1-Substituted 4 Tosylimidazoles”,Jour CuZZocrea, “Role of Peroxynitrite and Activation of Poly nal of Organic Chemistry, 47 (11):2043–6 (1982). (ADP-Ribose) Synthetase in the Vascular Failure Induced J. Wilson et al., “Local Anesthetics. Aminoalkoxyisoquino by Zymosan-activated Plasma’, Brit. J. Pharm., line Derivatives”, Journal of the American Chemical Soci 122:493-503 (1997). ety, vol. 71, pp. 937–938 (Mar. 1949). Endres et al., “Ischemic Brain Injury is Mediated by the H. Wright & M.B. Moore, “Local Anesthetics. V. 4-Mor Activation of Poly (ADP-Ribose) Polymerase,” J. Cerebral pholinylalkyl Aryl Ether”, Journal of the American Chemi Flood Flow Metabol., 17 (11): 1143–51 (1997). cal Society, vol. 76, pp. 4396–4398 (Sep. 5, 1954). Heller et al., “Inactivation of the Poly (ADP-Ribose) Poly Desilets et al., “Design and Synthesis of Near-Infrared merase Gene Affects Oxygen Radical and Nitric Oxide Absorbing Pigments”, Can. J. Chem., 73, 319-35 (1995). Toxicity in Islet Cells”, J. Biol. Chem., 270: 11176-80 (Part I and Part II). (1995). Langlois et al., “Synthesis of Quinazoline-2, 4-dione and Hughes et al., “Induction of T Helper Cell Hyporesponsive Naphthalimide Derivatives as New 5-HT3 Receptor ness in an Experimental Model of Autoimmunity by Using Antagonists”, Eur. J. Med. Chem., 29,925-40 (1994). Nonmitogenic Anti-CD3 Monoclonal Antibody”, J. Mao et al., “The inhibition of nitric oxide-activated poly (ADP-ribose) Synthetase attenuates transSynaptic alteration Immuno., 153:3319-25 (1994). of Spinal cord dorsal horn neurons and neuropathic pain in Salzman et al., “Role of Peroxynitrite and Poly (ADP-Ri the rat", Pain Vol. 72, pp. 355-366 (1997). bose) Synthase Activation Experimental Colitis”, Japanese Purnell et al., “Novel Inhibitors of Poly (ADP-Ribose) J. Pharm., 75, Supp. I:15 (1997). Synthase”, Biochem. J., 185, 775–77 (1980). Southan et al., “Spontaneous Rearrangement of Aminoalky Ruf et al., “Structure of the catalytic fragment of poly lisothioureas into Mercaptoalkylguanidines, a Novel Class (ADP-ribose) polymerase from chicken”, Proc. Natl. Acad. of Nitric Oxide Synthase Inhibitors with Selectivity Towards Sci. USA vol. 93, pp. 7481–7485 (Jul. 1996). the Inducible Isoform", Br. J. Pharm., 117:619-32 (1996). Vaziri et al., “ATM-dependent telomere loSS in aging Szabó et al., “DNA Strand Breakage, Activation of Poly human dipoloid fibroblasts and DNA damage lead to the (ADP-Ribose) Synthetase, and Cellular Energy Depletion post-translational activation of p53 protein involving poly are Involved in the Cytotoxicity in Macrophages and (ADP-ribose) polymerase”, The EMBO Journal vol. 16 No. Smooth Muscle Cells Exposed to Peroxynitrite', Proc. Natl. 19, pp. 6018–6033 (1997). Acad. Sci. USA, 93:1753–58 (1996). Thiemermann et al. Proc. Natl. Acad Sci. USA 94.679–83 Szabó et al., “Mercaptoethylguanidine and Guanidine 1997. Inhibitors of Nitric Oxide Synthase React with peroxynitrite Iadecola Trends Neurosci. 20:3, 132-139 1997. and Protect Against Peroxynitrite-induced Oxidative Dam Dawson et al. Cerebrovascular Disease 319-25 1997. age", J. Biol. Chem., 272:9030-36 (1997). White et al. Tetrahedron Letters 52:9, 3117–34 1996. Szabó et al., “Protective Effects of an Inhibitor of Poly Dawson et al. J. Neurosci. 16:8, 2479–87 1996. (ADP-Ribose) Synthetase in Collagen-Induced Arthritis,” Zhang et al. J. Neurochem 65:3, 1411–14 1995. Japanese J. Pharm., 75, Supp. I: 102 (1997). White et al. Tetrahedron Letters 36:33, 5983–86 1995. Wallis et al., Traumatic Neuroprotection with Inhibitors of R. Griffin et al. Anti-Cancer Drug Design 10(6): 507-14 Nitric Oxide and ADP-Ribosylation, Brain Res., 1995. 710:169–77 (1996). US 6,197,785 B1 Page 3

Weltin et al., “Effect of 6(5)-Phenanthridinone, and Inhibi Against Nitric Oxide-Mediated Delayed Vascular Failure tor of Poly (ADP-ribose) Polymerase, on Cultured Tumor in Endotoxic Shock: Potential Involvement of PolyADP Cells”, Oncol. Res., 6:399-403 (1994). Ribosyl Synthetase”, Shock, 5:258–64 (1996). Zingarelli et al., “Protective Effects of Nicontinamide * cited by examiner U.S. Patent Mar. 6, 2001 Sheet 1 of 2 US 6,197,785 B1

-O- CONTROL (n=14) -O- 10 mg/kg-treated group (n=7) 20

-as t

1 5

O

5

-2 O 2 4. 6 8 10 12 14 16 DISTANCE FROMINTERAURAL LINE (mm) F.G. 1 U.S. Patent Mar. 6, 2001 Sheet 2 of 2 US 6,197,785 B1

100

CONTROL 5mg/kg 10mg/kg 20mg/kg 40mg/kg (n=14) (n27) (n=7) (n=7) (n=7)

F.G. 2 US 6,197,785 B1 1 2 ALKOXY-SUBSTITUTED COMPOUNDS, (Endres et al., “Ischemic Brain Injury is Mediated by the METHODS, AND COMPOSITIONS FOR Activation of Poly(ADP-Ribose) Polymerase”, J. Cereb. INHIBITING PARPACTIVITY Blood Flow Metabol., 17:1143–51 (1997)) and in traumatic brain injury (Wallis et al., “Traumatic Neuroprotection with This application is a continuation-in-part application of 5 Inhibitors of Nitric Oxide and ADP-Ribosylation, Brain U.S. patent application Ser. No. 09/079,508, filed May 15, Res., 710:169–77 (1996)). 1998, now pending and also of U.S. patent application Ser. It has been demonstrated that single injections of PARP No. 08/922,520, filed Sep. 3, 1997, now abandoned the inhibitors have reduced the infarct Size caused by ischemia contents of which are incorporated herein in their entirety. and reperfusion of the heart or skeletal muscle in rabbits. In these studies, a single injection of the PARP inhibitor, BACKGROUND OF THE INVENTION 3-amino-benzamide (10 mg/kg), either one minute before 1. Field of the Invention occlusion or one minute before reperfusion, caused similar The present invention relates to inhibitors of the nucleic reductions in infarct size in the heart (32–42%). Another enzyme poly(adenosine 5'-diphospho-ribose) polymerase PARP inhibitor, 1,5-dihydroxyisoquinoline (1 mg/kg), "poly(ADP-ribose) polymerase” or “PARP”, which is also 15 reduced infarct size by a comparable degree (38-48%). sometimes called “PARS” for poly(ADP-ribose) Thiemermann et al., “Inhibition of the Activity of Poly(ADP Synthetase. More particularly, the invention relates to the Ribose) Synthetase Reduces Ischemia-Reperfusion Injury in use of PARP inhibitors to prevent and/or treat tissue damage the Heart and Skeletal Muscle', Proc. Natl. Acad. Sci. USA, resulting from cell damage or death due to necrosis or 94.679–83 (1997). This finding has suggested that PARP apoptosis, neural tissue damage resulting from ischemia and inhibitors might be able to Salvage previously ischemic heart reperfusion injury; neurological disorders and neurodegen or skeletal muscle tissue. erative diseases, to prevent or treat vascular Stroke; to treat PARP activation has also been shown to provide an index or prevent cardiovascular disorders, to treat other conditions of damage following neurotoxic insults by glutamate (via and/or disorderS Such as age-related macular degeneration, NMDA receptor stimulation), reactive oxygen 25 intermediates, amyloid B-protein, n-methyl-4-phenyl-1,2,3, AIDS and other immune Senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of 6-tetrahydropyridine (MPTP) and its active metabolite skeletal muscle involving replicative Senescence, diabetes, N-methyl-4-phenylpyridine (MPP), which participate in head trauma, immune Senescence, inflammatory bowel dis pathological conditions Such as Stroke, Alzheimer's disease orders (Such as colitis and Crohn's disease), muscular and Parkinson's disease. Zhang et al., “Poly(ADP-Ribose) dystrophy, Osteoarthritis, Osteoporosis, chronic and acute Synthetase Activation: An Early Indicator of Neurotoxic pain (Such as neuropathic pain), renal failure, retinal DNADamage”, J. Neurochem., 65:3, 1411–14 (1995). Other ischemia, Septic shock (Such as endotoxic shock), and skin studies have continued to explore the role of PARP activa aging, to extend the lifespan and proliferative capacity of tion in cerebellar granule cells in vitro and in MPTP neu cells, to alter gene expression of Senescent cells, or to rotoxicity. Cosi et al., “Poly(ADP-Ribose) Polymerase 35 (PARP) Revisited. A New Role for an Old Enzyme: PARP radioSensitize hypoxic tumor cells. Involvement in Neurodegeneration and PARP Inhibitors as 2. Description of the Prior Art Possible Neuroprotective Agents”, Ann. N.Y. Acad. Sci., Poly(ADP-ribose) polymerase (“PARP”) is an enzyme 825:366-79 (1997); and Cosi et al., “Poly(ADP-Ribose) located in the nuclei of cells of various organs, including Polymerase Inhibitors Protect Against MPTP-induced muscle, heart and brain cells. PARP plays a physiological 40 Depletions of Striatal Dopamine and Cortical Noradrenaline role in the repair of strand breaks in DNA. Once activated in C57B1/6 Mice", Brain Res., 729:264–69 (1996). by damaged DNA fragments, PARP catalyzes the attach Neural damage following Stroke and other neurodegen ment of up to 100 ADP-ribose units to a variety of nuclear erative processes is thought to result from a massive release proteins, including histones and PARP itself. While the exact of the excitatory neurotransmitter glutamate, which acts range of functions of PARP has not been fully established, 45 upon the N-methyl-D-aspartate (NMDA) receptors and this enzyme is thought to play a role in enhancing DNA other Subtype receptorS. Glutamate Serves as the predomi repair. nate excitatory neurotransmitter in the central nervous Sys During major cellular Stresses, however, the extensive tem (CNS). Neurons release glutamate in great quantities activation of PARP can rapidly lead to cell damage or death when they are deprived of oxygen, as may occur during an through depletion of energy stores. Four molecules of ATP 50 ischemic brain insult Such as a stroke or heart attack. This are consumed for every molecule of NAD (the source of exceSS release of glutamate in turn causes over-Stimulation ADP-ribose) regenerated. Thus, NAD, the substrate of (excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, PARP, is depleted by massive PARP activation and, in the Kainate and MGR receptors. When glutamate binds to these efforts to re-synthesize NAD, ATP may also be depleted. receptors, ion channels in the receptorS open, permitting It has been reported that PARP activation plays a key role 55 flows of ions across their cell membranes, e.g., Ca" and Na" in both NMDA- and NO-induced neurotoxicity, as shown by into the cells and K" out of the cells. These flows of ions, the use of PARP inhibitors to prevent such toxicity in especially the influx of Ca", cause overstimulation of the cortical cultures in proportion to their potencies as inhibitors neurons. The over-Stimulated neurons Secrete more of this enzyme (Zhang et al., “Nitric Oxide Activation of glutamate, creating a feedback loop or domino effect which Poly(ADP-Ribose) Synthetase in Neurotoxicity”, Science, 60 ultimately results in cell damage or death Via the production 263:687-89 (1994)); and in hippocampal slices (Wallis et of proteases, lipases and free radicals. Excessive activation al., “Neuroprotection Against Nitric Oxide Injury with of glutamate receptorS has been implicated in various neu Inhibitors of ADP-Ribosylation”, NeuroReport, 5:3, 245–48 rological diseases and conditions including epilepsy, Stroke, (1993)). The potential role of PARP inhibitors in treating Alzheimer's disease, Parkinson's disease, Amyotrophic Lat neurodegenerative diseases and head trauma has thus been 65 eral Sclerosis (ALS), Huntington's disease, Schizophrenia, known. Research, however, continues to pinpoint the exact chronic pain, ischemia and neuronal loSS following hypoxia, mechanisms of their Salutary effect in cerebral ischemia, hypoglycemia, ischemia, trauma, and nervous insult. Recent US 6,197,785 B1 3 4 Studies have also advanced a glutamatergic basis for com Carcino, and Muta., 16:219–27 (1996), discusses the effect pulsive disorders, particularly drug dependence. Evidence of hydrogen peroxide and Y-radiation on DNA Strand breaks includes findings in many animal Species, as well as, in in the presence of and in the absence of 3-aminobenzamide, cerebral cortical cultures treated with glutamate or NMDA, a potent inhibitor of PARP. Cristovao et al. observed a that glutamate receptor antagonists block neural damage PARP-dependent recovery of DNA strand breaks in leuko following vascular stroke. Dawson et al., “Protection of the cytes treated with hydrogen peroxide. Brain from Ischemia, Cerebrovascular Disease, 319-25 (H. Hunt Batjer ed., 1997). Attempts to prevent excitotox PARP inhibitors have been reported to be effective in icity by blocking NMDA, AMPA, Kainate and MGR recep radioSensitizing hypoxic tumor cells and effective in pre tors have proven difficult because each receptor has multiple venting tumor cells from recovering from potentially lethal Sites to which glutamate may bind. Many of the composi damage of DNA after radiation therapy, presumably by their tions that are effective in blocking the receptors are also ability to prevent DNA repair. See U.S. Pat. Nos. 5,032,617; toxic to animals. AS Such, there is no known effective 5,215,738; and 5,041,653. treatment for glutamate abnormalities. Evidence also exists that PARP inhibitors are useful for The stimulation of NMDA receptors, in turn, activates the treating inflammatory bowel disorders. Salzman et al., “Role enzyme neuronal nitric oxide synthase (NNOS), which 15 of Peroxynitrite and Poly(ADP-Ribose)Synthase Activation causes the formation of nitric oxide (NO), which more Experimental Colitis,” Japanese J. Pharm., 75, Supp. I: 15 directly mediates neurotoxicity. Protection against NMDA (1997), discusses the ability of PARP inhibitors to prevent or neurotoxicity has occurred following treatment with NOS treat colitis. Colitis was induced in rats by intraluminal inhibitors. See Dawson et al., "Nitric Oxide Mediates administration of the hapten trinitrobenzene Sulfonic acid in Glutamate Neurotoxicity in Primary Cortical Cultures”, 50% ethanol. Treated rats received 3-aminobenzamide, a Proc. Natl. Acad. Sci. USA, 88.6368–71 (1991); and Daw specific inhibitor of PARP activity. Inhibition of PARP Son et al., “Mechanisms of Nitric Oxide-mediated Neuro activity reduced the inflammatory response and restored the toxicity in Primary Brain Cultures”, J. NeuroSci., 13:6, morphology and the energetic Status of the distal colon. See also, Southan et al., “Spontaneous Rearrangement of Ami 2651–61 (1993). Protection against NMDA neurotoxicity 25 can also occur in cortical cultures from mice with targeted noalkylithioureas into Mercaptoalkylguanidines, a Novel disruption of NNOS. See Dawson et al., “Resistance to Class of Nitric Oxide Synthase Inhibitors with Selectivity Neurotoxicity in Cortical Cultures from Neuronal Nitric Towards the Inducible Isoform', Br. J. Pharm., 117:619–32 Oxide Synthase-Deficient Mice”, J. NeuroSci., 16:8, (1996); and Szabó et al., “Mercaptoethylguanidine and 2479–87 (1996). Guanidine Inhibitors of Nitric Oxide Synthase React with It is known that neural damage following vascular Stroke Peroxynitrite and Protect Against Peroxynitrite-induced is markedly diminished in animals treated with NOS inhibi Oxidative Damage”, J. Biol. Chem., 272:9030-36 (1997). tors or in mice with NNOS gene disruption. Iadecola, Evidence also exists that PARP inhibitors are useful for “Bright and Dark Sides of Nitric Oxide in Ischemic Brain treating arthritis. Szabó et al., “Protective Effects of an Injury”, Trends Neurosci., 20:3, 132-39 (1997); and Huang 35 Inhibitor of Poly(ADP-Ribose)Synthetase in Collagen et al., “Effects of Cerebral Ischemia in Mice Deficient in Induced Arthritis,” Japanese J. Pharm., 75, Supp. I: 102 Neuronal Nitric Oxide Synthase”, Science, 265:1883–85 (1997), discusses the ability of PARP inhibitors to prevent or (1994). See also, Beckman et al., “Pathological Implications treat collagen-induced arthritis. See also Szabó et al., “DNA of Nitric Oxide, Superoxide and Peroxynitrite Formation”, Strand Breakage, Activation of Poly(ADP-Ribose) Biochem. Soc. Trans., 21:330-34 (1993). Either NO or 40 Synthetase, and Cellular Energy Depletion are Involved in peroxynitrite can cause DNA damage, which activates the Cytotoxicity in Macrophages and Smooth Muscle Cells PARP. Further support for this is provided in Szabó et al., Exposed to Peroxynitrite,” Proc. Natl. Acad. Sci. USA, “DNA Strand Breakage, Activation of Poly(ADP-Ribose) 93: 1753–58 (March 1996); Bauer et al., “Modification of Synthetase, and Cellular Energy Depletion are Involved in Growth Related Enzymatic Pathways and Apparent Loss of the Cytotoxicity in Macrophages and Smooth Muscle Cells 45 Tumorigenicity of a ras-transformed Bovine Endothelial Exposed to Peroxynitrite”, Proc. Natl. Acad. Sci. USA, Cell Line by Treatment with 5-Iodo-6-amino-1,2- 93: 1753–58 (1996). benzopyrone (INHBP)”, Intl. J. Oncol., 8:239–52 (1996), Zhang et al., U.S. Pat. No. 5,587,384 issued Dec. 24, and Hughes et al., “Induction of T Helper Cell Hyporespon 1996, discusses the use of certain PARP inhibitors, Such as siveness in an Experimental Model of Autoimmunity by benzamide and 1,5-dihydroxy-isoquinoline, to prevent 50 Using Nonmitogenic Anti-CD3 Monoclonal Antibody”, J. NMDA-mediated neurotoxicity and, thus, treat stroke, Immuno., 153:3319-25 (1994). Alzheimer's disease, Parkinson's disease and Huntington's Further, PARP inhibitors appear to be useful for treating disease. However, it is has now been discovered that Zhang diabetes. Heller et al., “Inactivation of the Poly(ADP et al. may have been in error in classifying neurotoxicity as Ribose)Polymerase Gene Affects Oxygen Radical and Nitric NMDA-mediated neurotoxicity. Rather, it may have been 55 Oxide Toxicity in Islet Cells,” J. Biol. Chem., 270:19, more appropriate to classify the in Vivo neurotoxicity 11176-80 (May 1995), discusses the tendency of PARP to present as glutamate neurotoxicity. See Zhang et al. "Nitric deplete cellular NAD+ and induce the death of insulin Oxide Activation of Poly(ADP-Ribose) Synthetase in producing islet cells. Heller et al. used cells from mice with Neurotoxicity”, Science, 263:687-89 (1994). See also, Cosi inactivated PARP genes and found that these mutant cells et al., Poly(ADP-Ribose)Polymerase Inhibitors Protect 60 did not show NAD+ depletion after exposure to DNA Against MPTP-induced Depletions of Striatal Dopamine damaging radicals. The mutant cells were also found to be and Cortical Noradrenaline in C57B1/6 Mice', Brain Res., more resistant to the toxicity of NO. 729:264–69 (1996). Further still, PARP inhibitors have been shown to be It is also known that PARP inhibitors affect DNA repair useful for treating endotoxic Shock or Septic Shock. Zin generally. Cristovao et al., “Effect of a Poly(ADP-Ribose) 65 garelli et al., “Protective Effects of Nicotinamide Against Polymerase Inhibitor on DNA Breakage and Cytotoxicity Nitric Oxide-Mediated Delayed Vascular Failure in Endot Induced by Hydrogen Peroxide and Y-Radiation, Terato., oxic Shock: Potential Involvement of PolyADP Ribosyl US 6,197,785 B1 S 6 Synthetase,” Shock, 5:258-64 (1996), suggests that inhibi Accordingly, there remains a need for compounds that tion of the DNA repair cycle triggered by poly(ADPribose) inhibit PARP activity, compositions containing those com Synthetase has protective effects against Vascular failure in pounds and methods utilizing those compounds, wherein the endotoxic shock. Zingarelli et al. found that nicotinamide compounds produce more potent and reliable effects with protects against delayed, NO-mediated vascular failure in fewer side effects, with respect to inhibiting PARP activity endotoxic Shock. Zingarelli et al. also found that the actions of nicotinamide may be related to inhibition of the and treating the diseases and conditions discussed herein. NO-mediated activation of the energy-consuming DNA Further, other multicyclic, nitrogen-containing, alkoxy repair cycle, triggered by poly(ADPribose) Synthetase. See Substituted compounds are known: also, CuZZocrea, “Role of Peroxynitrite and Activation of Poly(ADP-Ribose) Synthetase in the Vascular Failure 1,3-Dihydro-4-methoxy-thieno 3,4-cquinoline, which Induced by Zymosan-activated Plasma, Brit. J. Pharm., has the following Structure: 122:493-503 (1997). Yet another known use for PARP inhibitors is treating OMe cancer. Suto et al., “Dihydroisoquinolinones: The Design 15 and Synthesis of a New Series of Potent Inhibitors of Poly(ADP-Ribose) Polymerase”, Anticancer Drug DeS., 7:107-17 (1991), discloses processes for synthesizing a number of different PARP inhibitors. In addition, Suto et al., U.S. Pat. No. 5,177,075, discusses several isoquinolines used for enhancing the lethal effects of ionizing radiation or chemotherapeutic agents on tumor cells. Weltin et al., “Effect of 6(5H)-Phenanthridinone, an Inhibitor of Poly (ADP-ribose) Polymerase, on Cultured Tumor Cells”, is disclosed in White et al., “Quinoline Analogues of Ortho Oncol. Res., 6:9,399–403 (1994), discusses the inhibition of 25 Quinodimethane.” Tetrahedron Letters, 36:33, 5983–86 PARP activity, reduced proliferation of tumor cells, and a (1995). This structure is also disclosed in White et al., marked Synergistic effect when tumor cells are co-treated “Dihydrothiophenes as Precursors to Fused Quinolines, Qui with an alkylating drug. no lone S and CoumarinS via O - Quinodime thane Still another use for PARP inhibitors is the treatment of Intermediates.” Tetrahedron Letters, 52.9, 3117–34 (1996). peripheral nerve injuries, and the resultant pathological pain Both of the White references also disclose the following Syndrome known as neuropathic pain, Such as that induced StructureS: by chronic constriction injury (CCI) of the common Sciatic nerve and in which transSynaptic alteration of spinal cord dorsal horn characterized by hyperchromatosis of cytoplasm OMe and nucleoplasm (so-called “dark” neurons) occurs. See 35 Mao et al., Pain, 72:355-366 (1997). PARP inhibitors have also been used to extend the OS lifespan and proliferative capacity of cells including treat ment of diseases Such as Skin aging, Alzheimer's disease, atherosclerosis, osteoarthritis, osteoporosis, muscular 40 dystrophy, degenerative diseases of Skeletal muscle involv ing replicative Sene Scence, age-related macular degeneration, immune Senescence, AIDS, and other immune It is not believed that the above disclosed compounds have Senescence diseases, and to alter gene expression of Senes 45 been shown to inhibit PARP activity per se. cent cells. See WO 98/27975. Large numbers of known PARP inhibitors have been SUMMARY OF THE INVENTION described in Banasik et al., “Specific Inhibitors of Poly (ADP-Ribose) Synthetase and Mono(ADP-Ribosyl)- The compounds of the present invention have formula I: Transferase”, J. Biol. Chem., 267:3, 1569–75 (1992), and in 50 Banasik et al., “Inhibitors and Activators of ADP I Ribosylation Reactions”, Molec. Cell. Biochem., 138:185-97 (1994). However, the approach of using these PARP inhibitors in the ways discussed above has been limited in effect. For 55 example, side effects have been observed with some of the best-known PARP inhibitors, as discussed in Milam et al., “Inhibitors of Poly(Adenosine Diphosphate-Ribose) Syn thesis: Effect on Other Metabolic Processes', Science, or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, 223:589-91 (1984). Specifically, the PARP inhibitors 60 prodrug, metabolite, Stereoisomer, or mixtures thereof, 3-aminobenzamide and benzamide not only inhibited the wherein: action of PARP but also were shown to affect cell viability, glucose metabolism, and DNA synthesis. Thus, it was con R", when present, is hydrogen or lower alkyl; cluded that the usefulness of these PARP inhibitors may be R is lower alkyl, aryl, aralkyl, lower alkanoyl, or severely restricted by the difficulty of finding a dose that will 65 -(CH2), (CHOH),(CH2)A, wherein n is 1-4, y is 0 inhibit the enzyme without producing additional metabolic or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower effects. alkanoyl, aryl, aralkyl, -NH, -NH-(lower alkyl), US 6,197,785 B1 7 8 R", when present, is hydrogen or lower alkyl; / lower alkwlyl) R is lower alkyl, aryl, aralkyl, lower alkanoyl, or -N -(CH), (CHOH),(CH), A, wherein n is 1-4, y is 0 (lower alkyl), or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower -O). -O) O alkanoyl, aryl, aralkyl, -NH, -NH-(lower alkyl), -O / lower alkwlyl) -N 1O (lower alkyl), -O). -O) O Y represents the atoms necessary to form a fused 5- to 6-membered ring that is aromatic or nonaromatic and carbocyclic or heterocyclic, Z is (i) -CHRCHR- where R and R are indepen -O dently hydrogen, alkyl, aryl or aralkyl, (ii) —RC=CR - where R and R are independently 15 hydrogen, lower alkyl, aryl, aralkyl, chlorine, bromine or Y represents the atoms necessary to form a fused 5- to -NR'R', where R and Rare independently hydrogen or 6-membered ring that is aromatic or nonaromatic and lower alkyl, or, R and R, taken together, form a fused 5 carbocyclic or heterocyclic, to 6-membered ring that is aromatic or nonaromatic and Z is (i) —CHRCHR- where R and R are indepen carbocyclic or heterocyclic, dently hydrogen, alkyl, aryl or aralkyl, (iii) -R°C=N-; (ii)—RC=CR- where R and Rare independently (iv) –CR(OH)-NR7-; or hydrogen, lower alkyl, aryl, aralkyl, chlorine, bro (v) -C(O)-NR'-; mine or -NR'R', where R7 and R are indepen provided that when R and R form a fused benzene ring, dently hydrogen or lower alkyl, or, RandR, taken then Y is neither (a) a fused, 6-membered, nonaromatic 25 together, form a fused 5- to 6-membered ring that is carbocyclic ring nor (b) a fused, 5-membered, nonaromatic aromatic or nonaromatic and carbocyclic or hetero heterocyclic ring having a Sulfur atom as its Sole heteroatom. cyclic; In another embodiment, a process of making the com (iii) -R°C=N-; pound of formula I: (iv) –CR(OH)-NR7-; or (v) -C(O)-NR'-; provided that when R and R form a fused benzene ring, then Y is neither (a) a fused, 6-membered, nonaromatic carbocyclic ring nor (b) a fused, 5-membered, nonaromatic heterocyclic ring having a Sulfur atom as its Sole heteroatom. 35 In particularly preferred embodiments of the above composition, the amount of the compound of formula I is present in an amount effective to inhibit PARP activity, to treat or prevent tissue damage regulting from cell damage or or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, death due to necrosis or apoptosis, to effect a neuronal prodrug, metabolite, Stereoisomer, or mixtures thereof, 40 activity not mediated by NMDA toxicity, to effect a neuronal wherein R', R, n, y, m, A, Y, Z, R, R, R', and R are as activity mediated by NMDA toxicity, to treat neural tissue defined above, comprises the Step of contacting an interme damage resulting from ischemia and reperfusion injury, diate having formula II: neurological disorders and neurodegenerative diseases, to prevent or treat vascular Stroke; to treat or prevent cardio I 45 vascular disorders, to treat other conditions and/or disorders Such as age-related macular degeneration, AIDS and other immune Senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative Senescence, diabetes, head trauma, 50 immune Senescence, inflammatory bowel disorders (Such as colitis and Crohn's disease), muscular dystrophy, with RX wherein X is a bromo, chloro or iodo moiety. osteoarthritis, osteoporosis, chronic and/or acute pain (Such In yet another embodiment, the pharmaceutical compo as neuropathic pain), renal failure, retinal ischemia, Septic Sition of the invention comprises a pharmaceutically accept Shock (Such as endotoxic shock), and skin aging, to extend 55 the lifespan and proliferative capacity of cells, to alter gene able carrier and a compound of formula I: expression of Senescent cells, or to radioSensitize hypoxic tumor cells.

In an additional embodiments, a method of inhibiting PARP activity comprises administering a compound of 60 formula I, as described above for the pharmaceutical com positions of the invention. In preferred embodiments, the amount of the compound administered in the methods of the invention is effective for treating tissue damage resulting from cell damage or death due to necrosis or apoptosis, or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, 65 neural tissue damage resulting from ischemia and reperfu prodrug, metabolite, Stereoisomer, or mixtures thereof, Sion injury, or neurological disorders and neurodegenerative wherein: diseases, to prevent or treat vascular Stroke, to treat or US 6,197,785 B1 9 10 prevent cardiovascular disorders, to treat other conditions prevent or treat vascular Stroke; to treat or prevent cardio and/or disorderS Such as age-related macular degeneration, vascular disorders, to treat other conditions and/or disorders AIDS and other immune Senescence diseases, arthritis, Such as age-related macular degeneration, AIDS and other atherosclerosis, cachexia, cancer, degenerative diseases of immune Senescence diseases, arthritis, atherosclerosis, skeletal muscle involving replicative Senescence, diabetes, head trauma, immune Senescence, inflammatory bowel dis cachexia, cancer, degenerative diseases of skeletal muscle orders (Such as colitis and Crohn's disease), muscular involving replicative Senescence, diabetes, head trauma, dystrophy, osteoarthritis, osteoporosis, chronic and/or acute immune Senescence, inflammatory bowel disorders (Such as pain (Such as neuropathic pain), renal failure, retinal colitis and Crohn's disease), muscular dystrophy, ischemia, Septic shock (Such as endotoxic shock), and skin osteoarthritis, osteoporosis, chronic and/or acute pain (Such aging, to extend the lifespan and proliferative capacity of as neuropathic pain), renal failure, retinal ischemia, Septic cells, to alter gene expression of Senescent cells, or to Shock (Such as endotoxic shock), and skin aging. radioSensitive hypoxic tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS Preferably, the compounds of the invention act as PARP 15 inhibitors to treat or prevent tissue damage resulting from FIG. 1 shows the distribution of the cross-sectional infarct area at representative levels along the rostrocaudal axis, as cell death or damage due to necrosis or apoptosis, to treat or measured from the interaural line in non-treated animals and prevent neural tissue damage resulting from cerebral in animals treated with 10 mg/kg of 3,4-dihydro-5-4-(1- ischemia and reperfusion injury or neurodegenerative dis piperidinyl)-butoxyl-1(2H)-isoquinolinone. eases in an animal; to extend and increase the lifespan and FIG. 2 shows the effect of intraperitoneal administration proliferative capacity of cells, to alter gene expression of of 3,4-dihydro-5-4-(1-piperidinyl)-butoxy-1(2H)- Senescent cells, and to radioSensitize tumor cells. These isoquinolinone on the infarct Volume. compounds are thought to interfere with more than the NMDA-neurotoxicity and NO-mediated biological path DETAILED DESCRIPTION OF THE 25 ways. Preferably, the compounds of the invention exhibit an INVENTION ICs for inhibiting PARP in vitro of about 100 uM or lower, The alkoxy-Substituted compounds of the present inven more preferably, about 25 uM or lower. tion inhibit PARP activity. As such, they may treat or prevent neural tissue damage resulting from cell damage or death due to necrosis or apoptosis, cerebral ischemia and reper The inventors have now discovered that select alkoxy fusion injury or neurodegenerative diseases in an animal; substituted compounds that inhibit PARP activity can ame they may extend the lifespan and proliferative capacity of liorate neural tissue damage, including that following focal cells and thus be used to treat or prevent diseases associated ischemia and reperfusion injury; can increase or extend the there with; they may alter gene expression of Senescent cells, 35 lifespan or proliferation of cells, can alter gene expression in and they may radioSensitize hypoxic tumor cells. Preferably, Senescent cells, and can radioSensitize tumor cells. the compounds of the invention treat or prevent tissue Generally, inhibition of PARP activity spares the cell from damage resulting from cell damage or death due to necrosis energy loSS, preventing irreversible depolarization of the or apoptosis, and/or effect neuronal activity, either mediated neurons and, thus, provides neuroprotection. While not or not mediated by NMDA toxicity. These compounds are 40 wishing to be bound thereby, it is thought that PARP thought to interfere with more than the glutamate neurotox icity and NO-mediated biological pathways. Further, the activation may play a common role in Still other excitotoxic compounds of the invention can treat or prevent other tissue mechanisms, perhaps as yet undiscovered, in addition to the damage related to PARP activation. production of free radicals and NO. Since PARP is necessary For example, the compounds of the invention can treat or 45 for DNA repair, the inhibition of PARP can also be used to prevent cardiovascular tissue damage resulting from cardiac prevent radiation damaged tumor cells from recovering from ischemia or reperfusion injury. Reperfusion injury, for potentially lethal damage of DNA by preventing DNA instance, occurs at the termination of cardiac bypass proce repair. PARP inhibitors may also be used to extend or dures or during cardiac arrest when the heart, once prevented increase the lifespan and proliferation of cells and to thus from receiving blood, begins to reperfuse. 50 prevent or treat diseases and conditions associated with The compounds of the present invention can also be used cellular Senescence, and can be used to alter the gene to extend or increase the lifespan or proliferation of cells and expression of Senescent cells can increase or extend the thus to treat or prevent diseases associated therewith and lifespan or proliferation of cells, can alter gene expression in induced or exacerbated by cellular Senescence including Senescent cells, and can radioSensitize tumor cells. skin aging, atherosclerosis, osteoarthritis, osteoporosis, 55 muscular dystrophy, degenerative diseases of Skeletal muscle involving replicative Senescence, age-related macu The compounds of the invention act as PARP inhibitors to lar degeneration, immune Senescence, AIDS and other treat or prevent neural tissue damage resulting from cerebral immune Senescence diseases, and other diseases associated ischemia and reperfusion injury or neuro-degenerative dis with cellular Senescence and aging, as well as to alter the 60 eases in an animal; to extend and increase the lifespan and gene expression of Senescent cells. These compounds can proliferative capacity of cells, to alter gene expression of also be used to treat cancer and to radioSensitize hypoxic Senescent cell; and to radioSensitize tumor cells. These tumor cells to render the tumor cells more Susceptible to compounds are thought to interfere with more than the radiation therapy and to prevent the tumor cells from recov NMDA-neurotoxicity and NO-mediated biological path ering from potentially lethal damage of DNA after radiation 65 ways. Preferably, the compounds of the invention exhibit an therapy, presumably by their ability to prevent DNA repair. ICs for inhibiting PARP in vitro of about 100 uM or lower, The compounds of the present invention can be used to more preferably, about 25 uM or lower. US 6,197,785 B1 11 12 The compound of the invention has the formula: tetrahydropyranyl, pyridyl, pyyrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl, oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridaZinyl, pyrimidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, thienyl, tetrahydro isoquinolinyl, cinnolinyl, phthala Zinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carboZolyl, acridinyl, phenazinyl, phenothiazinyl, phenox or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, azinyl and the like. prodrug, metabolite, Stereoisomer or mixtures thereof, Possible Substituents on an aryl group can be any non wherein Y represents the atoms necessary to form a fused 5 interfering Substituent. However, preferred Substituents to 6-membered ring that is aromatic or nonaromatic and include, without limitation, alkyl, alkenyl, alkoxy, phenoxy, carbocyclic or heterocyclic. benzyloxy, cycloalkyl, cycloalkenyl, hydroxy, carboxy, When Y forms a fused 5-membered carbocyclic ring, 15 carbonyl, amino, amido, cyano, isocyano, nitro, nitroSo, examples thereof include Such rings as fused cyclopentane, nitrilo, isonitrilo, imino, azo, diazo, Sulfonyl, Sulfoxy, thio, cyclopentene, cyclopentadiene rings and the like. When Y thiocarbonyl, sulfhydryl, halo, haloalkyl, trifluoro-methyl, forms a 5-membered N-containing heterocyclic ring, aryl and aralkyl. examples thereof include Such rings as fused pyrrole, R", when present, is hydrogen or lower alkyl. Examples isopyrrole, imidazole, isoimidazole, pyrazole, pyrrolidine, of useful alkyl groups are shown above as possible Substitu pyrroline, imidazolidine, imidazoline, pyrazolidine, ents for Y. pyrazoline, isothiazole, isoxazole, furazan, furan, thiophene, R is lower alkyl, aryl, aralkyl, lower alkanoyl, or 1,2,3-triazole, 1,2,4-triazole, dithiole, Oxathiole, isoxazole, -(CH2)(CHOH),(CH2)A wherein: Oxazole, thiazole, isothiazole, oxadiazole, Omatriazole, n is 1-4, preferably 1, dioxazole, Oxathiazole and the like rings. 25 When Y forms a fused 6-membered carbocyclic ring, y is 0 or 1, preferably 0; examples of useful Y groups include a fused cyclohexane, m is 0-5, preferably 0; and cyclohexene, benzene or the like, optionally Substituted with A is cycloalkyl, cycloalkenyl, lower alkanoyl, aryl, additional fused rings, thus forming, for example, aralkyl, -NH, -NH-(lower alkyl), naphthalene, anthracene, phenanthrene, benzonaphthene, and the like ring Systems. I Ikwl When Y forms a 6-membered N-containing heterocyclic ^ ower alkyl) ring, examples thereof include Such rings as pyridine, pyrazine, pyrimidine, pyridazine, piperidine, piperazine, Y(lower alkyl), -O). -O) O morpholine, pyran, pyrone, dioxin, triazine, oxazine, 35 isoxazine, Oxathiazine, oxadiazine, and the like rings. Y may be aromatic, Such as pyrrole, benzene or pyridine, -O or non-aromatic, Such as cyclopentene, piperidyl or piper azinyl. Preferably, Y has at least one site of unsaturation. wherein “alkyl”, “aryl” and “aralkyl” are as defined and/or Y may be unsubstituted or substituted with one or more 40 non-interfering Substituents. For example, Y may be Substi exemplified above. Examples of useful alkanoyl groups tuted with an alkyl group Such as methyl, ethyl, propyl, include formyl, acetyl, propanoyl, Sec-butanoyl, tert butyl, pentyl, hexyl, isopropyl, isobutyl, tert-butyl, n-pentyl, pentanoyl (2,2-dimethylpropanoyl), benzoyl, hexanoyl, 2-methylpentyl, 2-methylhexyl, dodecyl, octadecyl and the 3-methylpentanoyl, and the like. like, with an alkenyl group Such as ethenyl, propenyl, 45 When R is -(CH2)(CHOH),(CH2)A, examples of butenyl, pentenyl, 2-methylpentenyl, Vinyl, isopropenyl, useful groups include the following: 2,2-dimethyl-1-propenyl, decenyl, hexadecenyl and the like; with an alkynyl group Such as ethynyl, propynyl, butynyl, pentynyl, heXynyl, heptynyl, octynyl and the like, with an y A. alkanoyl group Such as formyl, acetyl, propanoyl, butanoyl, 50 pentanoyl, benzoyl and the like, with a cycloalkyl group 1. O O cyclohexyl 2 1. 1. cyclopentadienyl Such as adamantyl, cyclobutyl, cyclopentyl, cyclohexyl, 3 O 2 propanoyl cycloheptyl, cyclooctanyl, cyclononyl, cyclodecyl and the 4 1. 3 phenyl like, with a cycloalkenyl group Such as cyclopropenyl, 1. O 4 benzyl cyclopentadienyl, cyclohexenyl, cyclooctenyl and the like; 55 2 1. 5 -NH. with an aralkyl group Such as benzyl, 3-(1)-naphthyl-1- propyl, p-halobenzyl, p-ethylbenzyl, 1-phenyl-1-propyl, 3 O 1. McHs 3-pyridinyl-1-propyl, 1-phenyl-2-Sec-butyl, 4-phenyl-4- -N methyl-1-pentyl and the like; or with an aryl group Such as CHCH phenyl, naphthyl, pyridinyl, thienyl and the like. 60 "Aryl is defined as an unsaturated carbocyclic or het 4 1. 2 -NHCH, erocyclic moiety which may be either unsubstituted or Substituted with one or more non-interfering Substituent(s). 1. O 3 Examples include, without limitation, phenyl, naphthyl, / Y, indenyl, azulenyl, fluorenyl, anthracenyl, indolyl, isolindolyl, 65 \ / indolinyl, benzo-furanyl, benzothiophenyl, indazolyl, ben Zimidazolyl, ben Zithiazolyl, tetrahydrofurnayl, US 6,197,785 B1 13

-continued

y A. N 5 2 1. 4 O ON O O2 -N C N N isoquinoline phthalazine pteridine

3 O 5 1O i O ON O O O N N Preferred R groups include methyl, ethyl, propyl, butyl 15 quinazoline pyrido-bipyridine pyrido'-bipyridine and benzyl groups, with benzyl being particularly preferred. Z in formula I can be:

25 pyrazinodpyridazine purine phenanthridine Preferably, however, Z is -CHRCHR-, -RC=CR or-RC=N-. R° and R in formulas (i)-(v) above can be, or pharmaceutically acceptable Salt, hydrate, ester, Solvate, independently, hydrogen; alkyl, Such as methyl, ethyl, isopropyl, tert-butyl, n-pentyl, Sec-octyl, dodecyl and the prodrug, metabolite, Stereoisomer, or mixtures thereof. Pref like, aryl; or aralkyl. erably the compound of the invention has an isoquinoline, a In formula (ii) (-R'C=CR ), R and R, indepen phenanthridine, a phthalazine, a pteridine, or a quinazoline dently can be hydrogen, alkyl as described above, phenyl, nucleus. More preferably it has an isoquinoline or a phthala benzyl, chlorine, bromine, -NO, -COOH, -COOCH, Zine nucleus. or -NR'R''. When R is -NRR, R is independently 35 hydrogen or lower alkyl as described above. Preferred embodiments of the invention include Alternatively, RandR, taken together, can form a fused 1-(benzyloxy)-5-methylphthalazine (I), 1-(methoxy)-5- aromatic or non-aromatic, mono-, bi- or tricyclic, carbocy methylphthalazine (II), 1-(ethoxy)-5-methylphthalazine clic or heterocyclic ring, wherein each individual ring has 40 (III), 1-(propoxy)-5-methylphthalazine (IV), 1-(butoxy)-5- 5-6 ring member atoms. Examples of Such rings include a methylphthalazine (V), 1-(methoxy)-5-hydroxy-phthalazine fused cyclopentadiene, pyrrole, isopyrrole, imidazole, (VI), 1-(ethoxy)-5-hydroxy-phthalazine (VII), isoimidazole, triazole, pyrazole, pyridine, thiophene, furan, 1-(propoxyoxy)-5-hydroxyphthalazine (VIII), 1-(butoxy)-5- thiazole, isothiazole, oxazole, isoxazole, oxadiazole, benzene, naphthalene, acridine, cyclohexane, cyclohexene, hydroxyphthalazine (IX), 1-(benzyloxy)-5-methyl indole, pyran, pyrone, pyridine, pyrazine, pyrimidine, 45 isoquinoline (X), 1-(methoxy)-5-methylisoquinoline (XI), pyridazine, or triazine nucleus. When Z is -RC=CR 1-(ethoxy)-5-methylisoquinoline (XII), 1-(propoxy)-5- and forms a fused aromatic or non-aromatic ring, the ring methylisoquinoline (XIII), 1-(butoxy)-5-methylisoquinoline formed may be Substituted with one or more non-hydrogen (XIV), 1-(ethoxy)-5-hydroxyisoquinoline (XV), 1-(propoxy oxy)-5-hydroxyisoquinoline (XVI) and non-interfering Substituents. 50 Possible substituents of Y, or of the fused ring formed 1-(butoxy)-5-hydroxy-isoquinoline (XVII), the structures of when Zis-RC=CR , include any substituent that does which follow. not interfere with the reactions and purposes of the inven tion. Examples include, without limitation, C-C Straight or branched chain alkyl, C-Co Straight or branched chain alkenyl, C-C alkoxy, C-C, alkenyloxy, phenoxy, 55 benzyloxy, C-C cycloalkyl, Cs-C, cycloalkenyl, hydroxy, carboxy, carbonyl, amino, amido, cyano, isocyano, nitro, nitroSo, nitrilo, isonitrilo, imino, azo, diazo, Sulfonyl, Sulfoxy, thio, thiocarbonyl, Sulfhydryl, halo, haloalkyl, trifluoro-methyl, carbocyclic and heterocyclic moieties. Car 60 bocylic moieties include alicyclic and aromatic Structures. Preferably, however, Z is -RC=CR - where R and R, taken together, form a fused benzene ring, and neither Y nor Z is Substituted with a non-hydrogen Substituent. 65 In the compound of the invention, the multicyclic nuclear CH ring Structure is preferably one of the following: US 6,197,785 B1 15 16 -continued -continued VII II CH CH O s s 2N 2N

CH OH III VIII CH rt 15 O O

s n 2N N 2 N s CH OH IV 25 IX CH

O

n N 35 N 2 s

OH CH 40

45 O NN

s 2 CH 50 CH VI XI CH 55 l NN 2 60 CH

65 US 6,197,785 B1 17 -continued -continued XII XVI t CH O O NN s 21 NN 21 , and CH OH XIII 15 H XVII CH

O NN O 21 s n N 25 CH 21

XIV OH CH O Most preferred embodiments include 1-(benzyloxy)-5- NN methylphthalazine (I) and 1-(benzyloxy)-5-methyl isoquinoline (X), which are shown above. 2 s 35

OH Additional examples of useful inhibitors are shown below in Table I: XV

CH 40

n 45

s

-CH -CH-CH

US 6,197,785 B1 33 34

TABLE I-continued

OR2

-CH -CECH

Br

-CH2(CHS) -CECH

-CH -CECH NHH-CHs

-CH3(CHS) -CECH NHH CHs

-CECH -CCHs NH-CHs

-CH

-CH3(CHS)

ON -CCH 21

-CH N -CEN CH CH, CH

-CH2(CHS) N -CEN CH CH, CH3

US 6,197,785 B1 41 42

TABLE I-continued

-CCH ralsZ. -CH

OBr -CH2(CHS)

OBr

-CCHs

OBr -CH 21

N NO2 NH2

-CH3(CHS) 21

N

NO

21 -CCH N NO2 US 6,197,785 B1 43 44

TABLE I-continued

R2

-CH

-CH2(CHS) 21

CS

O| 21 -CCHs N

S

Also included are the pharmaceutically acceptable base or ammonia, alkali metal Salts. Such as Sodium, lithium and acid addition Salts, hydrate, ester, Solvate, prodrugs, potassium Salts, alkaline earth metal Salts Such as calcium metabolites, Stereoisomers, and mixtures thereof. and magnesium Salts, aluminum Salts, and Zinc Salts. The compounds of the invention may be useful in a free Salts, hydrates, esters, or Solvates may also be formed base form, in the form of pharmaceutically acceptable Salts, with organic bases. organic bases Suitable for the formation pharmaceutically acceptable hydrates, pharmaceutically of pharmaceutically acceptable base addition Salts, hydrates, acceptable esters, pharmaceutically acceptable Solvates, esters, or Solvates of the compounds of the present invention pharmaceutically acceptable prodrugs, pharmaceutically include those that are non-toxic and Strong enough to form acceptable metabolites, and in the form of pharmaceutically 45 Such Salts, hydrates, esters, or Solvates. For purposes of acceptable Stereoisomers. These forms are all within the illustration, the class of Such organic bases may include Scope of the invention. In practice, the use of these forms mono-, di-, and trialkylamines, Such as methylamine, amounts to use of the neutral compound. dimethylamine, triethylamine and dicyclohexylamine; “Pharmaceutically acceptable salt”, “hydrate”, “ester” or mono-, di- or trihydroxyalkylamines, Such as mono-, di-, “solvate” refers to a salt, hydrate, ester, or Solvate of the 50 and triethanolamine, amino acids, Such as arginine and inventive compounds which possesses the desired pharma lysine, guanidine, N-methyl-glucosamine, N-methyl cological activity and which is neither biologically nor glucamine; L-glutamine, N-methyl-piperazine, morpholine; otherwise undesirable. Organic acids can be used to produce ethylenediamine, N-benzyl-phenethylamine; (trihydroxy Salts, hydrates, esters, or Solvates Such as acetate, adipate, methyl)aminoethane; and the like. See, for example, “Phar alginate, a Spartate, benzoate, benzene Sulfonate, 55 maceutical Salts,” J. Pharm. Sci., 66:1, 1–19 (1977). p-tolueneSulfonate, bisulfate, Sulfanate, Sulfate, naphthylate, Accordingly, basic nitrogen-containing groups can be quat butyrate, citrate, camphorate, camphor Sulfonate, ernized with agents including: lower alkyl halides Such as cyclopentanepropionate, digluconate, dodecylsulfate, methyl, ethyl, propyl, and butyl chlorides, bromides and e thane Sulfonate, fumarate, glucoheptanoate, iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl glycerophosphate, he misulfate heptanoate, hexanoate, 60 and diamyl Sulfates, long chain halides Such as decyl, lauryl, 2-hydroxy ethane Sulfonate, lactate, male ate, myristyl and Stearyl chlorides, bromides and iodides, and methaneSulfonate, 2-naphthalenesulfonate, nicotinate, aralkyl halides Such as benzyl and phenethyl bromides. oxalate, tosylate and undecanoate. Inorganic acids can be The acid addition Salts, hydrates, esters, or Solvates of the used to produce Salts, hydrates, esters, or Solvates Such as basic compounds may be prepared either by dissolving the hydrochloride, hydrobromide, hydroiodide, and thiocyanate. 65 free base of a PARP inhibitor in an aqueous or an aqueous Examples of Suitable base Salts, hydrates, esters, or Sol alcohol Solution or other Suitable Solvent containing the Vates include hydroxides, carbonates, and bicarbonates of appropriate acid or base, and isolating the Salt by evaporat US 6,197,785 B1 45 46 ing the solution. Alternatively, the free base of the PARP are functionalization reactions, and generally consist of (1) inhibitor may be reacted with an acid, as well as reacting the oxidative and reductive reactions that alter and create new PARP inhibitor having an acid group thereon with a base, functional groups and (2) hydrolytic reactions that cleave Such that the reactions are in an organic Solvent, in which esters and amides to release masked functional groups. case the Salt Separates directly or can be obtained by These changes are usually in the direction of increased concentrating the Solution. polarity. "Pharmaceutically acceptable prodrug” refers to a deriva Phase II reactions are conjugation reactions in which the tive of the inventive compounds which undergoes biotrans drug, or often a metabolite of the drug, is coupled to an formation prior to exhibiting its pharmacological effect(s). endogenous Substrate, Such as glucuronic acid, acetic acid, The prodrug is formulated with the objective(s) of improved or Sulfuric acid. chemical Stability, improved patient acceptance and compliance, improved bioavailability, prolonged duration of TABLE II action, improved organ Selectivity, improved formulation Phase I Reactions (functionalization reactions): (e.g., increased hydroSolubility), and/or decreased side 15 (1) Oxidation via the hepatic microsomal P450 system: effects (e.g., toxicity). The prodrug can be readily prepared Aliphatic oxidation from the inventive compounds using methods known in the Aromatic hydroxylation art, such as those described by Burger's Medicinal Chem N-Dealkylation istry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, O-Dealkylation S-Dealkylation 949–982 (1995). For example, the inventive compounds can Epoxidation be transformed into prodrugs by converting one or more of Oxidative deamination the hydroxy or carboxy groups into esters. Sulfoxide formation “Pharmaceutically acceptable metabolite” refers to drugs Desulfuration N-Oxidation and N-hydroxylation that have undergone a metabolic transformation. After entry Dehalogenation into the body, most drugs are Substrates for chemical reac (2) Oxidation via nonmicrosomal mechanisms: tions that may change their physical properties and biologic 25 Alcohol and aldehyde oxidation effects. These metabolic conversions, which usually affect Purine oxidation the polarity of the compound, alter the way in which drugs Oxidative deamination (monoamine oxidase and diamine Oxidase) are distributed in and excreted from the body. However, in (3) Reduction: Some cases, metabolism of a drug is required for therapeutic AZO and nitro reduction effect. For example, anticancer drugs of the antimetabolite (4) Hydrolysis: Ester and amide hydrolysis class must be converted to their active forms after they have Peptide bond hydrolysis been transported into a cancer cell. Since must drugs Epoxide hydration undergo metabolic transformation of Some kind, the bio Phase II Reactions (conjugation reactions): chemical reactions that play a role in drug metabolism may 35 (1) Glucuronidation be numerous and diverse. The main Site of drug metabolism (2) Acetylation is the liver, although other tissues may also participate. (3) Mercapturic acid formation A feature characteristic of many of these transformations (4) Sulfate conjugation is that the metabolic products, or “metabolites', are more (5) N-, O-, and S-methylation polar than the parent drugs, although a polar drug does (6) Trans-sulfuration Sometimes yield a less polar product. Substances with high 40 lipid/water partition coefficients, which pass easily acroSS The compounds of the present invention possess one or membranes, also diffuse back readily from tubular urine more asymmetric center(s) and thus can be produced as through the renal tubular cells into the plasma. Thus, Such mixtures (racemic and non-racemic) of Stereoisomers, or as Substances tend to have a low renal clearance and a long individual R- and S-stereoisomers. The individual stereoi persistence in the body. If a drug is metabolized to a more 45 SomerS may be obtained by using an optically active Starting polar compound, one with a lower partition coefficient, its material, by resolving a racemic or non-racemic mixture of tubular reabsorption will be greatly reduced. Moreover, the an intermediate at Some appropriate Stage of Synthesis, or by Specific Secretory mechanisms for anions and cations in the resolving a compound of formula I. proximal renal tubules and in the parenchymal liver cells The term "isomers' refer to compounds having the same operate upon highly polar Substances. 50 number and kind of atoms, and hence, the same molecular As a specific example, phenacetin (acetophenetidin) and weight, but differing in respect to the arrangement or con acetanilide are both mild analgesic and antipyretic agents, figuration of the atoms. "Stereoisomers' are isomers that but are transformed within the body to a more polar and differ only in the arrangement of atoms in Space. "Enanti more effective metabolite, p-hydroxyacetanilid omers' are a pair of Stereoisomers that are non (acetaminophen), which is widely used today. When a dose 55 Superimposable mirror images of each other. “Diastereoiso of acetanilid is given to a perSon, the Successive metabolites mers' are Stereoisomers which are not mirror images of each peak and decay in the plasma Sequentially. During the first other. “Racemic mixture” means a mixture containing equal, hour, acetanilid is the principal plasma component. In the or roughly equal, parts of individual enantiomers. A “non Second hour, as the acetanilid level falls, the metabolite racemic mixture' is a mixture containing unequal, or Sub acetaminophen concentration reaches a peak. Finally, after a 60 Stantially unequal, parts of individual enantiomers or Stere few hours, the principal plasma component is a further oisomers. metabolite that is inert and can be excreted from the body. Synthesis of Compounds Thus, the plasma concentrations of one or more metabolites, Many nonalkoxy-substituted PARP inhibitors can be syn as well as the drug itself, can be pharmacologically impor thesized by known methods from Starting materials that are tant. 65 known, are themselves commercially available, or may be The reactions involved in drug metabolism are often prepared by Standard techniques of organic chemistry used classified into two groups, as shown in the Table II. Phase I to prepare corresponding compounds in the literature. See, US 6,197,785 B1 47 48 for example, Suto et al., “Dihydroisoquinolinones: The The above-described reaction can take place at varying Design and Synthesis of a New Series of Potent Inhibitors of temperatures depending, for example, upon the Solvent used, Poly(ADP-30 ribose) Polymerase”, Anticancer Drug DeS., the solubility of the intermediate of formula II and RX in 6:107-17 (1991), which discloses processes for synthesizing the Solvent being used, and the Susceptibility of the reactions a number of different PARP inhibitors. to oxidize or participate in Side reactions. Preferably, For example, the alkoxy-Substituted compounds of the invention may be prepared by reacting an alkoxide with a however, when the above reaction is used, it takes place at primary alkyl halide to yield an ether by an S2 pathway, a a temperature from about 0°C. to about 100° C., preferably process known as the Williamson ether Synthesis. at about room temperature. Specifically, an intermediate having formula II: The time required for the above reaction also can vary widely, depending on much the same factors. Typically, however, the reaction takes place within a time of about 5 minutes to about 24 hours, preferably from about 10 minutes to about two hours. The addition sequence of the intermediate of formula III, 15 the base, a solvent (if used), and the RX compound, can vary Significantly depending upon the relative reactivities of these materials, the purity of these materials, the temperature is reacted with RX, wherein X is bromo, chloro or iodo, as at which the reaction is performed, the degree of agitation shown below: II used in the reaction, and the like. Preferably, however, the intermediate of formula II is first dissolved in a solvent, the OH base is first added, and the RX compound is then added. The product, a compound of formula I, is isolated from N-R Y-Base the reaction mixture by conventional techniques, Such as by precipitating out, extraction with an immiscible Solvent 25 under appropriate pH conditions, evaporation, filtration, O crystallization, or by column chromatography on Silica gel and the like. Typically, however, the product is removed by Y Y- RX either crystallization or column chromatography on Silica gel. Precursor compounds can be prepared by methods known OR2 in the art. An intermediate of formula II may be prepared by contacting an intermediate having formula III: Y Y-R -- X

35

The alkoxide anion needed for the Williamson reaction to proceed is typically generated by reacting the compound of formula II with a strong base, Such as Sodium hydride, with the hydroxide of a Group I element, such as KOH or NaOH, KOH, KCO, NaCO, n-butyllithium or the like. 40 NaOH, to initiate a fusion reaction, for example, KOH The resulting acid-base reaction produces the intermediate fusion. The intermediate of formula III can also be prep anion for reaction with the halide RX. pared by other methods known in the art. See L. Paquette, More Specific examples of this reaction include: Principles of Modern Heterocyclic Chemistry, 273-307 (1968). Other variations and modifications of this invention using the synthetic pathways described above will be obvi ous to those skilled in the art. OH -ch-( ) Typically, the compounds of formula I used in the com SN C-HSCH.Bf NN Her position of the invention will have an ICso for inhibiting 21 KCO2-u3 21 poly(ADP-ribose) polymerase in vitro of 100 uM or lower, preferably 25 uM or lower, more preferably 12 uM or lower CH CH and, even more preferably, 12 mM or lower. Pharmaceutical Compositions A further aspect of the present invention is directed to a OH -ch-( ) pharmaceutical composition comprising a pharmaceutically SN CH-CHBr n N acceptable carrier or a diluent and a therapeutically effective He amount of a compound of formula I or a pharmaceutically N. K2CO3 2N acceptable Salt, prodrug, metabolite, Stereoisomer, or mix tures (hereafter, “a compound of formula I”). CH CH The formula I compounds of the invention are useful in 60 the manufacture of pharmaceutical formulations comprising Typically, the reaction shown above takes place in a an effective amount thereof in conjunction with or as an solvent that is inert with respect to both the intermediate of admixture with excipients or carrierS Suitable for either formula II or RX, and allows at least some of the RX to enteral or parenteral application. AS Such, formulations of go into Solution. Typical Solvents include, for example, the present invention Suitable for oral administration may be methylene chloride, chloroform, tetrahydrofuran, 65 in the form of discrete units Such as capsules, cachets, dimethylformamide, and a variety of other inert organic tablets, troche or lozenges, each containing a predetermined Solvents. amount of the active ingredient; in the form of a powder or US 6,197,785 B1 49 SO granules, in the form of a Solution or a Suspension in an including olive oil and castor oil, especially in their poly aqueous liquid or nonaqueous liquid; or in the form of an oxyethylated forms, are also useful in the preparation of oil-in-water emulsion or a water-in-oil emulsion. The active injectables. These oil Solutions or Suspensions may also ingredient may also be in the form of a bolus, electuary, or contain long-chain alcohol diluents or dispersants. paste. Sterile Saline is a preferred carrier, and the compounds are The composition will usually be formulated into a unit often Sufficiently water Soluble to be made up as a Solution dosage form, Such as a tablet, capsule, aqueous Suspension for all foreseeable needs. The carrier may contain minor or Solution. Such formulations typically include a Solid, amounts of additives, Such as Substances that enhance Semisolid, or liquid carrier. Exemplary carriers include Solubility, isotonicity, and chemical Stability, e.g., anti lactose, dextrose, Sucrose, Sorbitol, mannitol, Starches, gum oxidants, buffers and preservatives. acacia, calcium phosphate, mineral oil, cocoa butter, oil of When administered rectally, the composition will usually theobroma, alginates, tragacanth, gelatin, Syrup, methyl be formulated into a unit dosage form Such as a Suppository cellulose, polyoxyethylene Sorbitan monolaurate, methyl or cachet. These compositions can be prepared by mixing hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium the compound with Suitable non-irritating excipients that are Stearate, corn Starch and the like. 15 Solid at room temperature, but liquid at rectal temperature, Particularly preferred formulations include tablets and Such that they will melt in the rectum to release the com gelatin capsules comprising the active ingredient together pound. common excipients include cocoa butter, beeswax with (a) diluents, Such as lactose, dextrose, Sucrose, and polyethylene glycols or other fatty emulsions or SuS mannitol, Sorbitol, cellulose, dried corn Starch, and glycine; pensions. and/or (b) lubricants, such as Silica, talcum, Stearic acid, its Moreover, the compounds may be administered topically, magnesium or calcium Salt, and polyethylene glycol. especially when the conditions addressed for treatment Tablets may also contain binders, Such as magnesium involve areas or organs readily accessible by topical aluminum Silicate, Starch paste, gelatin, tragacanth, application, including neurological disorders of the eye, the methylcellulose, Sodium carboxymethylcellulose and poly skin or the lower intestinal tract. Vinylpyrrolidone; disintegrants, Such as Starches, agar, alg 25 For topical application to the eye, or ophthalmic use, the inic acid or its Sodium Salt, and effervescent mixtures, and/or compounds can be formulated as micronized Suspensions in absorbents, colorants, flavors, and Sweeteners. The compo isotonic, pH-adjusted Sterile Saline or, preferably, as a Solu Sitions of the invention may be Sterilized and/or contain tion in isotonic, pH-adjusted Sterile Saline, either with or adjuvants, Such as preserving, Stabilizing, Swelling or emul without a preservative Such as benzylalkonium chloride. Sifying agents, Solution promoters, Salts for regulating Alternatively, the compounds may be formulated into oSmotic pressure, and/or buffers. In addition, the composi ointments, Such as petrolatum. tion may also contain other therapeutically valuable Sub For topical application to the skin, the compounds can be Stances. Aqueous Suspensions may contain emulsifying and formulated into Suitable ointments containing the com Suspending agents combined with the active ingredient. All pounds Suspended or dissolved in, for example, mixtures oral dosage forms may further contain Sweetening and/or 35 with one or more of the following: mineral oil, liquid flavoring and/or coloring agents. petrolatum, white petrolatum, propylene glycol, polyoxy These compositions are prepared according to conven ethylene compound, polyoxypropylene compound, emulsi tional mixing, granulating, or coating methods, respectively, fying wax and water. Alternatively, the compounds can be and contain about 0.1 to 75% of the active ingredient, formulated into Suitable lotions or creams containing the preferably about 1 to 50% of the same. tablet may be made 40 active compound Suspended or dissolved in, for example, a by compressing or molding the active ingredient optionally mixture of one or more of the following: mineral oil, with one or more accessory ingredients. Compressed tablets Sorbitan monoStearate, polySorbate 60, cetyl ester wax, may be prepared by compressing, in a Suitable machine, the cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and active ingredient in a free-flowing form Such as a powder or Water. granules, optionally mixed with a binder, lubricant, inert 45 Topical application to the lower intestinal tract can be diluent, Surface active, or dispersing agent. Molded tablets effected in rectal Suppository formulations (See above) or in may be made by molding, in a Suitable machine, a mixture Suitable enema formulations. of the powdered active ingredient and a Suitable carrier Formulations Suitable for nasal or buccal administration, moistened with an inert liquid diluent. (Such as Self-propelling powder dispensing formulations), When administered parenterally, the composition will 50 may comprise about 0.1% to about 5% w/w of the active normally be in a unit dosage, Sterile injectable form ingredient or, for example, about 1% W/w of the same. In (aqueous isotonic Solution, Suspension or emulsion) with a addition, Some formulations can be compounded into a pharmaceutically acceptable carrier. Such carriers are pref Sublingual troche or lozenge. erably non-toxic, parenterally-acceptable and contain non The formulations may conveniently be presented in unit therapeutic diluents or Solvents. Examples of Such carriers 55 dosage form and may be prepared by any of the methods include water; acqueous Solutions, Such as Saline (isotonic well known in the art of pharmacy. All methods include the Sodium chloride Solution), Ringer's Solution, dextrose Step of bringing the active ingredient into association with Solution, and Hanks Solution; and nonaqueous carriers, Such the carrier which constitutes one or more accessory ingre as 1,3-butanediol, fixed oils (e.g., corn, cottonseed, peanut, dients. In general, the formulations are prepared by uni Sesame oil, and Synthetic mono- or di-glyceride), ethyl 60 formly and intimately bringing the active ingredient into oleate, and isopropyl myristate. asSociation with a liquid carrier or a finely divided Solid Oleaginous Suspensions can be formulated according to carrier or both, and then, if necessary, Shaping the product techniques known in the art using Suitable dispersing or into the desired formulation. Wetting agents and Suspending agents. Among the accept In a preferred embodiment, the carrier is a Solid biode able Solvents or Suspending mediums are Sterile fixed oils. 65 gradable polymer or mixture of biodegradable polymers For this purpose, any bland fixed oil may be used. Fatty with appropriate time release characteristics and release acids, Such as oleic acid and its glyceride derivatives, kinetics. The composition of the invention may then be US 6,197,785 B1 S1 52 molded into a Solid implant Suitable for providing effica limitation, neurons, neural Support cells, glia, Schwann cious concentrations of the compounds of the invention over cells, vasculature contained within and Supplying these a prolonged period of time without the need for frequent Structures, the central nervous System, the brain, the brain re-dosing. The composition of the present invention can be stem, the Spinal cord, the junction of the central nervous incorporated into the biodegradable polymer or polymer System with the peripheral nervous System, the peripheral mixture in any Suitable manner known to one of ordinary nervous System, and allied Structures. skill in the art and may form a homogeneous matrix with the The term “ischemia' refers to localized tissue anemia due biodegradable polymer, or may be encapsulated in Some to obstruction of the inflow of arterial blood. Global ischemia occurs when blood flow to the entire brain ceases way within the polymer, or may be molded into a Solid for a period of time. Global ischemia may result from implant. In one embodiment, the biodegradable polymer or cardiac arrest. Focal ischemia occurs when a portion of the polymer mixture is used to form a Soft “depot' containing brain is deprived of its normal blood Supply. Focal ischemia the pharmaceutical composition of the present invention that may result from thromboembolytic occlusion of a cerebral can be administered as a flowable liquid, for example, by vessel, traumatic head injury, edema or brain tumor. Even if injection, but which remains Sufficiently Viscous to maintain transient, both global and focal ischemia can cause wide the pharmaceutical composition within the localized area 15 Spread neuronal damage. Although nerve tissue damage around the injection site. The degradation time of the depot occurs over hours or even days following the onset of So formed can be varied from Several days to a few years, ischemia, Some permanent nerve tissue damage may depending upon the polymer Selected and its molecular develop in the initial minutes following the cessation of weight. By using a polymer composition in injectable form, blood flow to the brain. Much of this damage has been even the need to make an incision may be eliminated. In any attributed to glutamate toxicity and to the Secondary conse event, a flexible or flowable delivery “depot' will adjust to quences of tissue reperfusion, Such as the release of vaso the shape of the Space it occupies within the body with a active products by damaged endothelium and the release of minimum of trauma to Surrounding tissues. The pharmaceu cytotoxic products, Such as free radicals and leukotrines, by tical composition of the present invention is used in amounts the damaged tissue. Ischemia can also occur in the heart in that are therapeutically effective, and may depend upon the 25 myocardial infarction and other cardiovascular disorders in desired release profile, the concentration of the pharmaceu which the coronary arteries have been obstructed as a result tical composition required for the Sensitizing effect, and the of atherosclerosis, thrombi, or Spasm and in the eyes in length of time that the pharmaceutical composition has to be retinal ischemia. released for treatment. The term “neural tissue damage resulting from ischemia The composition of the invention is preferably adminis and reperfusion injury and neurodegenerative diseases tered as a capsule or tablet containing a single or divided includes neurotoxicity, Such as Seen in vascular Stroke and dose of the compound, or as a Sterile Solution, Suspension, global and focal ischemia, as well as retinal ischemia. or emulsion, for parenteral administration in a single or The term “neurodegenerative diseases” includes Alzhe divided dose. imer's disease, Parkinson's disease and Huntington's dis In another preferred embodiment, the compounds of the 35 CSC. invention can be prepared in lyophilized form. In this case, The term “nervous insult” refers to any damage to ner 1 to 100 mg of a PARP inhibitor may be lyophilized in Vous tissue and any disability or death resulting therefrom. individual Vials, together with a carrier and a buffer, Such as The cause of nervous insult may be metabolic, toxic, mannitol and Sodium phosphate. The composition may then neurotoxic, iatrogenic, thermal or chemical, and includes be reconstituted in the vials with bacteriostatic water before 40 without limitation, ischemia, hypoxia, cerebrovascular administration. accident, trauma, Surgery, pressure, mass effect, The compounds of the invention are used in the compo hemmorrhage, radiation, vasospasm, neurodegenerative sition in amounts that are therapeutically effective. While the disease, infection, Parkinson's disease, amyotrophic lateral effective amount of the PARP inhibitor will depend upon the Sclerosis (ALS), myelination/demyelination process, particular compound being used, amounts of the these 45 epilepsy, cognitive disorder, glutamate abnormality and Sec compounds varying from about 1% to about 65% have been ondary effects thereof. easily incorporated into liquid or Solid carrier delivery The term “neuroprotective” refers to the effect of Systems. reducing, arresting or ameliorating nervous insult, and Compositions and Methods for Effecting Neuronal Activity protecting, resuscitating, or reviving nervous tissue that has Preferably, according to the invention, an effective thera 50 Suffered nervous insult. peutic amount of the compounds and compositions The term “preventing neurodegeneration' includes the described above are administered to animals to effect a ability to prevent neurodegeneration in patients diagnosed neuronal activity, preferably one that is not mediated by with a neurodegenerative disease or who are at risk of NMDA neurotoxicity. Such neuronal activity may consist of developing a neurodegenerative disease. The term also Stimulation of damaged neurons, promotion of neuronal 55 encompasses preventing further neurodegeneration in regeneration, prevention of neurodegeneration and treat patients who are already Suffering from or have Symptoms of ment of a neurological disorder. Accordingly, the present a neurodegenerative disease. invention further relates to a method of effecting a neuronal The term “treating” refers to: activity in an animal, comprising administering an effective (i) preventing a disease, disorder or condition from occur amount of the compound of formula I to Said animal. 60 ring in an animal that may be predisposed to the Further, the compounds of the invention inhibit PARP activ disease, disorder and/or condition, but has not yet been ity and, thus, are believed to be useful for treating neural diagnosed as having it; tissue damage, particularly damage resulting from cerebral (ii) inhibiting the disease, disorder or condition, i.e., ischemia and reperfusion injury or neurodegenerative dis arresting its development, and eases in animals. 65 (iii) relieving the disease, disorder or condition, i.e., The term “nervous tissue” refers to the various compo causing regression of the disease, disorder and/or con nents that make up the nervous System including, without dition. US 6,197,785 B1 S3 S4 Examples of neurological disorders that are treatable by disease), muscular dystrophy, osteoarthritis, osteoporosis, the method of using the present invention include, without chronic and/or acute pain (Such as neuropathic pain), renal limitation, trigeminal neuralgia; gloSSopharyngeal neural failure, retinal ischemia, Septic shock (Such as endotoxic gia, Bell's Palsy, myasthenia gravis, muscular dystrophy; Shock), and skin aging, to extend the lifespan and prolifera amyotrophic lateral Sclerosis, progressive muscular atrophy; tive capacity of cells, to alter gene expression of Senescent progressive bulbar inherited muscular atrophy; herniated, cells, or to radioSensitize tumor cells ruptured or prolapsed invertebrate disk Syndromes, cervical Further still, the methods of the invention can be used to spondylosis, plexus disorders, thoracic outlet destruction treat cancer and to radioSensitize tumor cells. The term Syndromes, peripheral neuropathies Such as those caused by “cancer' is interpreted broadly. The compounds of the lead, dapsone, ticks, porphyria, or Guillain-Barré Syndrome; present invention can be “anti-cancer agents', which term Alzheimer's disease, Huntington's Disease and Parkinson's also encompasses “anti-tumor cell growth agents' and “anti disease. neoplastic agents'. For example, the methods of the inven The method of the present invention is particularly useful tion are useful for treating cancers and radioSensitizing for treating a neurological disorder Selected from the group tumor cells in cancerS Such as ACTH-producing tumors, consisting of peripheral neuropathy caused by physical 15 acute lymphocytic leukemia, acute nonlymphocytic injury or disease State; head trauma, Such as traumatic brain leukemia, cancer of the adrenal cortex, bladder cancer, brain injury; physical damage to the Spinal cord; Stroke associated cancer, breast cancer, cervical cancer, chronic lymphocytic with brain damage, Such as vascular Stroke associated with leukemia, chronic myelocytic leukemia, colorectal cancer, hypoxia and brain damage, focal cerebral ischemia, global cutaneous T-cell lymphoma, endometrial cancer, esophageal cerebral ischemia, and cerebral reperfusion injury; demyeli cancer, Ewing's Sarcoma, gallbladder cancer, hairy cell nating diseases, Such as multiple Sclerosis, and neurological leukemia, head & neck cancer, Hodgkin's lymphoma, Kapo disorders related to neurodegeneration, Such as Alzheimer's Si’s Sarcoma, kidney cancer, liver cancer, lung cancer (Small Disease, Parkinson's Disease, Huntington's Disease and and/or non-Small cell), malignant peritoneal effusion, malig amyotrophic lateral Sclerosis (ALS). nant pleural effusion, melanoma, mesothelioma, multiple Treating Other PARP-Related Disorders 25 myeloma, neuroblastoma, non-Hodgkin's lymphoma, The compounds, compositions and methods of the present Osteosarcoma, ovarian cancer, ovary (germ cell) cancer, invention are particularly useful for treating or preventing prostate cancer, pancreatic cancer, penile cancer, tissue damage resulting from cell death or damage due to retinoblastoma, skin cancer, Soft-tissue Sarcoma, Squamous necrosis or apoptosis. cell carcinomas, Stomach cancer, testicular cancer, thyroid The compounds, compositions and methods of the inven cancer, trophoblastic neoplasms, uterine cancer, vaginal tion can also be used to treat a cardiovascular disorder in an cancer, cancer of the Vulva and Wilm's tumor. animal, by administering an effective amount of the com The term “radioSensitizer”, as used herein, is defined as pound of formula to the animal. molecule, preferably a low molecular weight molecule, AS used herein, the term "cardiovascular disorders' refers administered to animals in therapeutically effective amounts to those disorders that can either cause ischemia or are 35 to increase the Sensitivity of the cells to be radioSensitized to caused by reperfusion of the heart. Examples include, but electromagnetic radiation and/or to promote the treatment of are not limited to, coronary artery disease, angina pectoris, diseases which are treatable with electromagnetic radiation. myocardial infarction, cardiovascular tissue damage caused Diseases which are treatable with electromagnetic radiation by cardiac arrest, cardiovascular tissue damage caused by include neoplastic diseases, benign and malignant tumors, cardiac bypass, cardiogenic Shock, and related conditions 40 and cancerous cells. Electromagnetic radiation treatment of that would be known by those of ordinary skill in the art or other diseases not listed herein are also contemplated by the which involve dysfunction of or tissue damage to the heart present invention. The terms “electromagnetic radiation” or vasculature, especially, but not limited to, tissue damage and “radiation” as used herein includes, but is not limited to, related to PARP activation. radiation having the wavelength of 10° to 10” meters. For example, the methods of the invention are believed to 45 Preferred embodiments of the present invention employ the be useful for treating cardiac tissue damage, particularly electromagnetic radiation of gamma-radiation (10 to damage resulting from cardiac ischemia or caused by rep 10 m) X-ray radiation (10 to 10 m), ultraviolet light erfusion injury in animals. The methods of the invention are (10 nm to 400 nm), visible light (400 nm to 700 nm), particularly useful for treating cardiovascular disorders infrared radiation (700 nm to 1.0 mm), and microwave Selected from the group consisting of coronary artery 50 radiation (1 mm to 30 cm). disease, Such as atherOSclerosis, angina pectoris, myocardial RadioSensitizers are known to increase the Sensitivity of infarction; myocardial ischemia and cardiac arrest, cardiac cancerous cells to the toxic effects of electromagnetic radia bypass, and cardiogenic shock. The methods of the inven tion. Several mechanisms for the mode of action of radi tion are particularly helpful in treating the acute forms of the oSensitizers have been Suggested in the literature including: above cardiovascular disorders. 55 hypoxic cell radiosensitizers (e.g., 2-nitro-imidazole Further, the methods of the invention can be used to treat compounds, and benzotriazine dioxide compounds) promote tissue damage resulting from cell damage or death due to the reoxygenation of hypoxic tissue and/or catalyze the necrosis or apoptosis, neural tissue damage resulting from generation of damaging oxygen radicals; non-hypoxic cell ischemia and reperfusion injury, neurological disorders and radiosensitizers (e.g., halogenated pyrimidines) can be ana neurodegenerative diseases, to prevent or treat vascular 60 logs of DNA bases and preferentially incorporate into the Stroke; to treat or prevent cardiovascular disorders, to treat DNA of cancer cells and thereby promote the radiation other conditions and/or disorderS Such as age-related macu induced breaking of DNA molecules and/or prevent the lar degeneration, AIDS and other immune Senescence normal DNA repair mechanisms, and various other potential diseases, arthritis, atherosclerosis, cachexia, cancer, degen mechanisms of action have been hypothesized for radiosen erative diseases of skeletal muscle involving replicative 65 Sitizers in the treatment of disease. Senescence, diabetes, head trauma, immune Senescence, Many cancer treatment protocols currently employ radi inflammatory bowel disorders (such as colitis and Crohn's oSensitizers activated by the electromagnetic radiation of US 6,197,785 B1 SS S6 X-rayS. Examples of X-ray activated radioSensitizers include, In So proceeding, the physician or veterinarian may but are not limited to, the following: metronidazole, employ an intravenous bolus followed by an intravenous misonidazole, desmethylmisonidazole, pimonidazole, infusion and repeated administrations, as considered appro etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, priate. In the methods of the present invention, the com EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine pounds may be administered, for example, orally, (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, parenterally, by inhalation Spray, topically, rectally, nasally, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and buccally, Sublingually, vaginally, intraventricularly, or via an therapeutically effective analogs and derivatives of the implanted reservoir in dosage formulations containing con SC. ventional non-toxic pharmaceutically-acceptable carriers, Photodynamic therapy (PDT) of cancers employs visible 1O adjuvants and vehicles. light as the radiation activator of the Sensitizing agent. Parenteral includes, but is not limited to, the following Examples of photodynamic radioSensitizers include the examples of administration: intravenous, Subcutaneous, following, but are not limited to: hematoporphyrin intramuscular, intraspinal, intraosseous, intraperitoneal, derivatives, Photofrin, benzoporphyrin derivatives, NPe6, intrathecal, intraventricular, intrasternal or intracranial injec tin etio porphyrin Sn ET2, p he ob orbide -a, 15 tion and infusion techniques, Such as by Subdural pump. bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, Invasive techniques are preferred, particularly direct admin Zinc phthalocyanine, and therapeutically effective analogs istration to damaged neuronal tissue. While it is possible for and derivatives of the Same. the compound of formula I to be administered alone, it is RadioSensitizers may be administered in conjunction with preferable to provide it as a part of a pharmaceutical a therapeutically effective amount of one or more other formulation. compounds, including but not limited to: compounds which To be effective therapeutically as central nervous System promote the incorporation of radioSensitizers to the target targets, the compounds used in the methods of the present cells, compounds which control the flow of therapeutics, to invention should readily penetrate the blood-brain barrier nutrients, and/or oxygen to the target cells, chemotherapeu when peripherally administered. Compounds which cannot tic agents which act on the tumor with or without additional 25 penetrate the blood-brain barrier, however, can still be radiation; or other therapeutically effective compounds for effectively administered by an intraventricular route. treating cancer or other disease. Examples of additional The compounds used in the methods of the present therapeutic agents that may be used in conjunction with invention may be administered by a single dose, multiple radioSensitizers include, but are not limited to: discrete doses or continuous infusion. Since the compounds 5-fluorouracil, leucovorin, 5'-amino-5'deoxythymidine, are small, easily diffusible and relatively stable, they are well oxygen, carbogen, red cell transfusions, perfluorocarbons Suited to continuous infusion. Pump means, particularly (e.g., Fluosol-DA), 2,3-DPG, BW12C, calcium channel Subcutaneous or Subdural pump means, are preferred for blockers, pentoxyfylline, antiangiogenesis compounds, continuous infusion. hydralazine, and L-BSO. Examples of chemotherapeutic For the methods of the present invention, any effective agents that may be used in conjunction with radioSensitizers 35 administration regimen regulating the timing and Sequence include, but are not limited to: adriamycin, camptothecin, of doses may be used. Doses of the compounds preferably carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, include pharmaceutical dosage units comprising an effica interferon (alpha, beta, gamma), interleukin 2, irinotecan, cious quantity of active compound. By an efficacious quan paclitaxel, topotecan, and therapeutically effective analogs tity is meant a quantity sufficient to inhibit PARP activity and derivatives of the Same. 40 and/or derive the desired beneficial effects therefrom The compounds of the present invention may also be used through administration of one or more of the pharmaceutical for radioSensitizing tumor cells. dosage units. In a particularly preferred embodiment, the The term “treating” refers to: dose is Sufficient to prevent or reduce the effects of Vascular (i) preventing a disease, disorder or condition from occur Stroke or other neurodegenerative diseases. ring in an animal that may be predisposed to the 45 An exemplary daily dosage unit for a vertebrate host disease, disorder and/or condition, but has not yet been comprises an amount of from about 0.001 mg/kg to about 50 diagnosed as having it; mg/kg. Typically, dosage levels on the order of about 0.1 mg (ii) inhibiting the disease, disorder or condition, i.e., to about 10,000 mg of the active ingredient compound are arresting its development, and useful in the treatment of the above conditions, with pre (iii) relieving the disease, disorder or condition, i.e., 50 ferred levels being about 0.1 mg to about 1,000 mg. The causing regression of the disease, disorder and/or con Specific dose level for any particular patient will vary dition. depending upon a variety of factors, including the activity of Administration the Specific compound employed; the age, body weight, For medical use, the amount required of a compound of general health, Sex, and diet of the patient; the time of formula I to achieve a therapeutic effect will vary according 55 administration; the rate of excretion; any combination of the to the particular compound administered, the route of compound with other drugs; the Severity of the particular administration, the animal under treatment, and the particu disease being treated; and the form and route of adminis lar disorder or disease concerned. A Suitable Systemic dose tration. Typically, in vitro dosage-effect results provide of a compound of formula I for an animal Suffering from, or useful guidance on the proper doses for patient administra likely to Suffer from, any condition as described herein is 60 tion. Studies in animal models can also be helpful. The typically in the range of about 0.1 to about 100 mg of base considerations for determining the proper dose levels are per kilogram of body weight, preferably from about 1 to well-known in the art. about 10 mg/kg of animal body weight. It is understood that In methods of treating nervous insult (particularly acute the ordinarily skilled physician or veterinarian will readily ischemic Stroke and global ischemia caused by drowning or be able to determine and prescribe the amount of the 65 head trauma), the compounds of the invention can be compound effective for the desired prophylactic or thera co-administered with one or more other therapeutic agents, peutic treatment. preferably agents which can reduce the risk of Stroke (Such US 6,197,785 B1 57 58 as aspirin) and, more preferably, agents which can reduce the ery of the compound can be used and repeated as necessary risk of a second ischemic event (Such as ticlopidine). to effect treatment. Such regimen may include pretreatment The compounds and compositions can be co-administered and/or co-administration with additional therapeutic agents. with one or more therapeutic agents either (i) together in a To maximize protection of nervous tissue from nervous Single formulation, or (ii) Separately in individual formula insult, the compounds of the invention should be adminis tions designed for optimal release rates of their respective tered to the affected cells as Soon as possible. In Situations active agent. Each formulation may contain from about where nervous insult is anticipated, the compounds are 0.01% to about 99.99% by weight, preferably from about advantageously administered before the expected nervous 3.5% to about 60% by weight, of the compound of the insult. Such situations of increased likelihood of nervous invention, as well as one or more pharmaceutical excipients, insult include Surgery, Such as carotid endarterectomy, Such as wetting, emulsifying and pH buffering agents. When cardiac, Vascular, aortic, orthopedic Surgery; endovascular the compounds used in the methods of the invention are procedures, Such as arterial catheterization (carotid, administered in combination with one or more other thera vertebral, aortic, cardia, renal, spinal, Adamkiewicz); injec peutic agents, Specific dose levels for those agents will tions of embolic agents, the use of coils or balloons for depend upon considerations Such as those identified above hemostasis, interruptions of vascularity for treatment of for compositions and methods of the invention in general. 15 brain lesions, and predisposing medical conditions Such as For example, Table III below provides known median crescendo transient ischemic attacks, emboli and Sequential dosages for Selected chemotherapeutic agents that may be Strokes. administered in combination with the compounds of the Where pre-treatment for stroke or ischemia is impossible invention to Such diseases or various cancers. or impracticable, it is important to bring the compounds of the invention into contact with the affected cells as Soon as TABLE III possible, either during or after the event. In the time period between Strokes, however, diagnosis and treatment proce CHEMOTHERAPEUTICAGENT MEDIAN DOSAGE dures should be minimized to save the cells from further damage and death. Therefore, a particularly advantageous Asparaginase 10,000 units Bleomycin Sulfate 15 units 25 mode of administration with a patient diagnosed with acute Carboplatin 50-450 mg multiple vascular Strokes is by implantation of a Subdural Carmustine 100 mg pump to deliver the compounds of the invention directly to Cisplatin 10-50 mg the infarct area of the brain. Even if comatose, it is expected Cladribine 10 mg that the patient would recover more quickly than he or she Cyclophosphamide (lyophilized) 100 mg to 2 gm Cyclophosphamide (non-lyophilized) 100 mg to 2 gm would without this treatment. Moreover, in any conscious Cytarabine (lyophilized powder) 100 mg to 2 gm State of the patient, it is expected that any residual neuro Dacarbazine 100-200 mg logical Symptoms, as well as the re-occurrence of Stroke, Dactinomycin 0.5 mg would be reduced. Daunorubicin 20 mg As to patients diagnosed with other acute disorders Diethylstilbestrol 250 mg believed to be related to PARP activity, such as diabetes, Doxorubicin 10-150 mg 35 Etidronate 300 mg arthritis and Crohn's disease, the compound of the invention Etoposide 100 mg should also be administered as Soon as possible in a Single Floxuridine 500 mg or divided dose. Fludarabine Phosphate 50 mg Depending on the patient's presenting Symptoms and the Fluorouracil 500 mg to 5 gm Goserelin 3.6 mg degree of response to the initial administration of the com Granisetron Hydrochloride 1 mg 40 pound of the invention, the patient may further receive Idarubicin 5-10 mg additional doses of the same or different compounds of the Ifosfamide 1-3 gm Leucovorin Calcium 50-350 mg invention, by one of the following routes: parenterally, Such Leuprolide 3.75-7.5 mg as by injection or by intravenous administration, orally, Such Mechlorethamine 10 mg as by capsule or tablet, by implantation of a biocompatible, Medroxyprogesterone 1 gm 45 biodegradable polymeric matrix delivery System comprising Melphalan 50 gm Methotrexate 20 mg to 1 gm the compound; or by direct administration to the infarct area Mitomycin 5-40 mg by insertion of a Subdural pump or a central line. It is Mitoxantrone 20-30 mg expected that the treatment would alleviate the disorder, Ondansetron Hydrochloride 40 mg either in part or in its entirety and that fewer further Paclitaxel 30 mg 50 Pamidronate Disodium 30-90 mg occurrences of the disorder would develop. It also is Pegaspargase 750 units expected that the patient would Suffer fewer residual Symp Plicamycin 2,500 mcgm tomS. Streptozocin 1 gm Where a patient is diagnosed with an acute disorder prior Thiotepa 15 mg to the availability of the compounds of the invention, the Teniposide 50 mg 55 Vinblastine 10 mg patient's condition may deteriorate due to the acute disorder Vincristine 1-5 mg and become a chronic disorder by the time that the com Aldesleukin 22 million units pounds are available. Even when a patient receives a com Epoetin Alfa 2,000-10,000 units Filgrastim 300-480 mcgm pound of formula I for the chronic disorder, it is also Immune Globulin 500 mg to 10 gm expected that the patient's condition would stabilize and Interferon Alpha-2a 3-36 million units 60 actually improve as a result of receiving the compound. The Interferon Alpha-2b 3-50 million units compounds of the present invention may also be used to Levamisole 50 mg Octreotide 1,000-5,000 mcgm prevent disorders by prophylactic administration of the Sargramostim 250-500 mcgm compounds of the present invention. 65 EXAMPLES For the methods of the present invention, any adminis The following examples are illustrative of preferred tration regimen regulating the timing and Sequence of deliv embodiments of the invention or related inventions and are US 6,197,785 B1 59 60 not to be construed as limiting the present invention thereto. All polymer molecular weights are mean average molecular -continued weights. All percentages are based on the percent by weight of the final delivery System or formulation prepared unless PARP Inhibitor Approximate ICso's otherwise indicated, and all totals equal 100% by weight.

Example 1 Approximate ICso Data for Selected Compounds 3.4 uM The ICs of with respect to PARP inhibition was deter mined for several compounds by a PARP assay using purified recombinant human PARP from Trevigen (Gaithersburg, Md.), as follows: The PARP enzyme assay was set up on ice in a Volume of 100 microliters consisting of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl, 28 mM KC1, 28 mM NaCl, 0.1 mg/ml of herring sperm DNA (activated as a 15 1 mg/ml stock for 10 minutes in a 0.15% hydrogen peroxide solution), 3.0 micromolar 3Hnicotinamide adenine 50 uM dinucleotide (470 mci/mmole), 7 micrograms/ml PARP enzyme, and various concentrations of the compounds to be tested. The reaction was initiated by incubating the mixture at 25 C. After 15 minutes incubation, the reaction was terminated by adding 500 microliters of ice cold 20% (w/v) trichloroacetic acid. The precipitate formed was transferred onto a glass fiber filter (Packard Unifilter-GF/B) and washed 0.8 uM three times with ethanol. After the filter was dried, the 25 radioactivity was determined by Scintillation counting. Using the PARPassay described above, approximate ICso values were obtained for the following compounds:

COOCH PARP Inhibitor Approximate ICsors O NH2 .25 uM COOH 35 NH

40 NH2 O 1.6 uM 100 uM NH

21 45

NO

O 1.3 uM 50

NH 2 0.9 uM 55

NH O 10 uM

N 60

NO2 CCOOH US 6,197,785 B1 61 62 The rats were then anesthetized with halothane (4% for -continued induction and 0.8%-1.2% for the surgical procedure) in a mixture of 70% nitrous oxide and 30% oxygen. The body PARP Inhibitor Approximate ICsors temperature was monitored by a rectal probe and maintained O 5.2 uM at 37.5+0.5 C. with a heating blanket regulated by a homeothermic blanket control unit (Harvard Apparatus Limited, Kent, U.K.). A catheter (PE-50) was placed into the tail artery, and arterial pressure was continuously monitored and recorded on a Grass polygraph recorder (Model 7D, Grass Instruments, Quincy, Mass.). Samples for blood gas analysis (arterial pH, PaC) and PaCO) were also taken from the tail artery catheter and measured with a blood gas C. analyzer (ABL 30, Radiometer, Copenhagen, Denmark). O 0.7 uM Arterial blood samples were obtained 30 minutes after MCA 15 occlusion. The head of the animal was positioned in a Stereotaxic frame, and a right parietal incision between the right lateral canthus and the external auditory meatus was made. Using a dental drill constantly cooled with saline, a 3 mm burr hole was prepared over the corteX Supplied by the right MCA, 4 mm lateral to the Sagittal Suture and 5 mm caudal to the coronal Suture. The dura mater and a thin inner bone layer C were kept, care being taken to position the probe over a C. tissue area devoid of large blood vessels. The flow probe (tip O 0.2 uM 25 diameter of 1 mm, fiber separation of 0.25 mm) was lowered to the bottom of the cranial burr hole using a micromanipu lator. The probe was held stationary by a probe holder Secured to the Skull with dental cement. The microvascular blood flow in the right parietal cortex was continuously monitored with a laser Doppler flowmeter (FloLab, Moor, Devon, U.K., and Periflux 4001, Perimed, Stockholm, Sweden). Focal cerebral ischemia was produced by cauterization of C.F the distal portion of the right MCA with bilateral temporary 35 common carotid artery (CCA) occlusion by the procedure of O 1.1 uM Chen et al., “A Model of Focal Ischemic Stroke in the Rat: Reproducible Extensive Cortical Infarction”, Stroke 17:738–43 (1986) and/or Liu et al., “Polyethylene Glycol conjugated Superoxide Dismutase and Catalase Reduce 40 Ischemic Brain Injury”, Am. J. Physiol. 256:H589–93 (1989), both of which are hereby incorporated by reference. Specifically, bilateral CCA's were isolated, and loops made from polyethylene (PE-10) catheter were carefully Br passed around the CCA's for later remote occlusion. The C. 45 incision made previously for placement of the laser doppler probe was extended to allow observation of the rostral end Similar ICs values are obtained for the alkoxy-substituted of the Zygomatic arch at the fusion point using a dental drill, compounds of the invention. and the dura mater overlying the MCA was cut. The MCA Example 2 distal to its crossing with the inferior cerebral vein was lifted Neuroprotective Effect of DPO on Focal Cerebral Ischemia 50 by a fine StainleSS Steel hook attached to a micromanipulator in Rats and, following bilateral CCA occlusion, the MCA was Focal cerebral ischemia was produced by cauterization of cauterized with an electrocoagulator. The burr hole was the right distal MCA (middle cerebral artery) with bilateral covered with a Small piece of Gelform, and the wound was temporary common carotid artery occlusion in male Long Sutured to maintain the brain temperature within the normal Evans rats for 90 minutes. All procedures performed on the animals were approved by the University Institutional Ani 55 or near-normal range. mal Care and Use Committee of the University of Pennsyl After 90 minutes of occlusion, the carotid loops were vania. A total of 42 rats (weights: 230-340 g) obtained from released, the tail arterial catheter was removed, and all of the Charles River were used in this study. The animals fasted wounds were Sutured. Gentamicin Sulfate (10 mg/ml) was overnight with free access to water prior to the Surgical topically applied to the wounds to prevent infection. The procedure. 60 anesthetic was discontinued, and the animal was returned to Two hours prior to MCA occlusion, varying amounts his cage after awakening. Water and food were allowed ad (control, n=14, 5 mg/kg, n=7; 10 mg/kg, n=7; 20 mg/kg, libitum. n=7; and 40 mg/kg, n=7) of the compound, 3,4-dihydro-5- Two hours after MCA occlusion, the animals were given 4-(1-piperidinyl)-butoxy-1(2H)-isoquinolinone (“DPQ') the same doses of the PARP inhibitor as in the pre-treatment. were dissolved in dimethyl sulfoxide (DMSO) using a 65 Twenty-four hours after MCA occlusion, the rats were Sonicator. A volume of 1.28 ml/kg of the resulting Solution Sacrificed with an intraperitoneal injection of pentobarbital was injected intraperitoneally into fourteen rats. sodium (150 mg/kg). The brain was carefully removed from US 6,197,785 B1 63 64 the skull and cooled in ice-cold artificial CSF for five The values of arterial blood gases (PaC), PaCO and pH) minutes. The cooled brain was then Sectioned in the coronal were within the physiological range in the control and plane at 2 mm intervals using a rodent brain matrix (RBM treated groups with no significant differences in these 4000C, ASI Instruments, Warren, Mich.). The brain slices parameters among the five groups, as shown below in Table were incubated in phosphate-buffered Saline containing 2% IV. A “steady state' MABP was taken following completion 2,3,5-triphenyltetrazolium chloride (TTC) at 37° C. for ten of the Surgical preparation, just prior to occlusion; an minutes. Color photographs were taken of the posterior “ischemia' MABP was taken as the average MABP during Surface of the Stained slices and were used to determine the occlusion. See Table III below: damaged area at each cross-sectional level using a computer-based image analyzer (NIH Image 1.59). To avoid TABLE IV artifacts due to edema, the damaged area was calculated by Subtracting the area of the normal tissue in the hemisphere MABP (mm Hg) ipsilateral to the Stroke from the area of the hemisphere PaO, PaCO, Steady Ischemia contralateral to the Stroke, by the method of Swanson et al., (mm Hg) (mm Hg) pH State “A Semiautomated Method for Measuring Brain Infarct Control group 25 - 21 38.6 4.6 7.33 + 79 - 14 91 13 Volume”, J. Cereb. Blood Flow Metabol. 10:290–93 (1990), 15 (n = 4) O.05 the disclosure of which is hereby incorporated by reference. 5 mg/kg-treated 126 - 20 38.0 + 2.8 7.36 it 78 5 91 12 group (n = 7) O.O2 The total volume of infarction was calculated by Summation 10 mg/kg- 25 - 16 39.3 + 5.2 7.34 it 80 - 9 90 - 14 of the damaged Volume of the brain Slices. treated group O.O3 The cauterization of the distal portion of the right MCA (n = 7) with bilateral temporary CCA occlusion consistently pro 20 mg/kg- 22 - 14 41.3 + 2.8 7.35 - 79 - 10 91 + 12 duced a well-recognized cortical infarct in the right MCA treated group O.23 (n = 7) territory of each test animal. There was an apparent unifor 40 mg/kg- 37 - 17 39.5 47 7.33 78 + 4 88 - 12 mity in the distribution of the damaged area as measured by treated group O.24 TTC Staining in each group, as shown in FIG. 1. (n = 7) In FIG. 1, the distribution of the cross-sectional infarct 25 area at representative levels along the rostrocaudal axis was * = Significantly different from the steady state measured from the interaural line in non-treated animals and ** = Significantly different from the steady state value, p < 0.01. in animals treated with 10 mg/kg of 3,4-dihydro-5-4-(1- There were no significant differences in any physiological piperidinyl)-butoxy-1(2H)-isoquinolinone. The area of parameter, including mean arterial blood pressure (MABP), damage was expressed as meant Standard deviation. Signifi prior to MCA and CCA occlusion among the five groups. cant differences between the 10 mg-treated group and the Although MABP was significantly elevated following occlu control group were indicated (p<0.02, p<0.01, p<0.001). Sion in all five groups, there were no significant differences The 5 mg/kg and 20 mg/kg curves fell approximately in MABP during the occlusion period among the groups. halfway between the control and the 10 mg/kg curves, Since the blood flow values obtained from the laser whereas the 40 mg/kg curve was close to the control. The 5, 35 doppler were in arbitrary units, only percent changes from 20 and 40 mg/kg curves were omitted for clarity. the baseline (prior to occlusion) were reported. Right MCA PARP inhibition led to a significant decrease in the and bilateral CCA occlusion produced a significant decrease damaged volume in the 5 mg/kg-treated group (106.7t23.2 in relative blood flow in the right parietal cortex to mm, p<0.001), the 10 mg/kg-treated group (76.4+16.8 20.8+7.7% of the baseline in the control group (n=5), mm, p<0.001), and the 20 mg/kg-treated group (110.2:42.0 40 18.7+7.4% in the 5 mg/kg-treated group (n=7), 21.4+7.7% mm, p<0.01), compared to the control group (165.2+34.0 in the 10 mg/kg-treated group (n=7) and 19.3+11.2% in the mm). The data are expressed as meanistandard deviation. 40 mg/kg-treated group (n=7). There were no significant The Significance of differences between groups was deter differences in the blood flow response to occlusion among mined using an analysis of variance (ANOVA) followed by the four groups. In addition, blood flow showed no signifi 45 cant changes throughout the entire occlusion period in any Student's t-test for individual comparisons. grOup. There was no significant difference between the control Following release of the carotid occlusions, a good recov and the 40 mg/kg-treated group (135.6+44.8 mm). ery of blood flow (sometimes hyperemia) was observed in However, there were significant differences between the 5 the right MCA territory of all animals. Reperfusion of the mg/kg-treated group and the 10 mg/kg-treated group ischemic tissue resulted in the formation of NO and (p<0.02), and between the 10 mg/kg-treated group and the 50 40 mg/kg-treated group (p<0.01), as shown in FIG. 2. peroxynitrite, in addition to oxygen-derived free radicals. In FIG. 2, the effect of intraperitoneal administration of All of these radicals have been shown to cause DNA strand 3,4-dihydro-5-4-(1-pipe ridinyl)-butoxy-1(2H)- breaks and to activate PARP, This example provided evidence that the related com isoaquinolinone on the infarct Volume was depicted graphi pounds of the present invention are effective in inhibiting cally. The Volumes of infarct were expressed as 55 meantStandard deviation. Significant differences between PARP activity. the treated groupS and the control group were indicated Example 3 (p<0.01, "p-0.001). It is not clear why a high dose (40 Assay for Neuroprotective Effects on Focal Cerebral mg/kg) of the PARP inhibitor, 3,4-dihydro-5-4-(1- Ischemia in Rats piperidinyl)-butoxy-1(2H)-isoquinolinone, was less neuro 60 Focal cerebral ischemia experiments are performed using protective. The U-shaped dose-response curve may Suggest male Wistar rats weighing 250-300 g, which are anesthe dual effects of the compound. tized with 4% halothane. Anesthesia is maintained with However, overall, the in vivo administration of the inhibi 1.0-1.5% halothane until the end of Surgery. The animals are tor led to a Substantial reduction in infarct Volume in the installed in a warm environment to avoid a decrease in body focal cerebral ischemia model in the rat. This result indi 65 temperature during Surgery. cated that the activation of PARP plays an important role in An anterior midline cervical incision is made. The right the pathogenesis of brain damage in cerebral ischemia. common carotid artery (CCA) is exposed and isolated from US 6,197,785 B1 65 66 the vagus nerve. A Silk Suture is placed and tied around the Various doses of the compounds of the invention are CCA in proximity to the heart. The external carotid artery tested in this model. The compounds are given either in a (ECA) is then exposed and ligated with a silk Suture. A Single dose or a Series of multiple doses, i.p. or i.V., at puncture is made in the CCA and a small catheter (PE 10, different times, both before or after the onset of ischemia. Ulrich & Co., St-Gallen, Switzerland) is gently advanced to The compounds of the invention are found to have ischemia/ the lumen of the internal carotid artery (ICA). The ptery reperfusion injury protection in the range of 10 to 40 gopalatine artery is not occluded. The catheter is tied in percent. Therefore, they protect against ischemia-induced place with a silk Suture. Then, a 4-0 nylon Suture (Braun degeneration of rat hippocampal neurons in Vitro. Medical, Crissier, Switzerland) is introduced into the cath eter lumen and is pushed until the tip blocks the anterior Example 5 cerebral artery. The length of catheter into the ICA is Retinal Ischemia Protection approximately 19 mm from the origin of the ECA. The A patient just diagnosed with acute retinal ischemia is Suture is maintained in this position by occlusion of the immediately administered parenterally, either by intermit catheter with heat. One cm of catheter and nylon Suture are left protruding so that the suture can be withdrawn to allow tent or continuous intravenous administration, a compound reperfusion. The skin incision is then closed with wound 15 of Formula I, either as a single dose or a Series of divided clips. doses of the compound. After this initial treatment, and The animals are maintained in a warm environment depending on the patient's presenting neurological during recovery from anesthesia. Two hours later, the ani Symptoms, the patient optionally may receive the same or a mals are re-anesthetized, the clips are discarded, and the different compound of the invention in the form of another wound is re-opened. The catheter is cut, and the Suture is parenteral dose. It is expected by the inventors that Signifi pulled out. The catheter is then obturated again by heat, and cant prevention of neural tissue damage would ensue and wound clips are placed on the wound. The animals are that the patient's neurological Symptoms would consider allowed to Survive for 24 hours with free access to food and ably lessen due to the administration of the compound, water. The rats are then Sacrificed with CO and decapitated. leaving fewer residual neurological effects post-Stroke. In The brains are immediately removed, frozen on dry ice 25 addition, it is expected that the re-occurrence of retinal and stored at -80 C. The brains are then cut in 0.02 ischemia would be prevented or reduced. mm-thick Sections in a cryocut at -19 C., Selecting one of every 20 sections for further examination. The selected Example 6 Sections are Stained with cresyl Violet according to the Nissl Treatment of Retinal Ischemia procedure. Each Stained Section is examined under a light A patient has just been diagnosed with acute retinal microScope, and the regional infarct area is determined ischemia. Immediately, a physician or a nurse parenterally according to the presence of cells with morphological administers a compound of Formula I, either as a single dose changes. or as a Series of divided doses. The patient also receives the Various doses of the compounds of the invention are same or a different PARP inhibitor by intermittent or con tested in this model. The compounds are administered in tinuous administration via implantation of a biocompatible, either a single dose or a Series of multiple doses, i.p. or i.v., 35 biodegradable polymeric matrix delivery System comprising at different times, both before or after the onset of ischemia. a compound of Formula I, or via a Subdural pump inserted Compounds of the invention are found to provide protection to administer the compound directly to the infarct area of the from ischemia in the range of about 20 to 80%. brain. It is expected by the inventors that the patient would Example 4 awaken from the coma more quickly than if the compound Effects on Heart Ischemia/Reperfusion Iniury in Rats 40 of the invention were not administered. The treatment is also Female Sprague-Dawley rats, each weighing about expected to reduce the Severity of the patient's residual 300-350 g are anesthetized with intraperitoneal ketamine at neurological Symptoms. In addition, it is expected that a dose of 150 mg/kg. The rats are endotracheally intubated re-occurrence of retinal ischemia would be reduced. and ventilated with oxygen-enriched room air using a Har vard rodent ventilator. Polyethylene catheters inserted into 45 Example 7 the carotid artery and the femoral vein are used for artery Vascular Stroke Protection blood pressure monitoring and fluid administration respec A patient just diagnosed with acute vascular Stroke is tively. Arterial pCO is maintained between 35 and 45 mm immediately administered parenterally, either by intermit Hg by adjusting the respirator rate. The ratchests are opened tent or continuous intravenous administration, a compound by median Sternotomy, the pericardium is incised, and the 50 of Formula I, either as a single dose or a Series of divided hearts are cradled with a latex membrane tent. Hemody doses of the compound. After this initial treatment, and namic data are obtained at baseline after at least a 15-minute depending on the patient's presenting neurological Stabilization period following the end of the Surgical opera Symptoms, the patient optionally may receive the same or a tion. The LAD (left anterior descending) coronary artery is different compound of the invention in the form of another ligated for 40 minutes, and then re-perfused for 120 minutes. 55 parenteral dose. It is expected by the inventors that Signifi After 120 minutes reperfusion, the LAD artery is cant prevention of neural tissue damage would ensue and re-occluded, and a 0.1 ml bolus of monastral blue dye is that the patient's neurological Symptoms would consider injected into the left atrium to determine the ischemic risk ably lessen due to the administration of the compound, region. leaving fewer residual neurological effects post-Stroke. In The hearts are then arrested with potassium chloride and 60 addition, it is expected that the re-occurrence of vascular cut into five 2-3 mm thick transverse slices. Each Slice is stroke would be prevented or reduced. weighed and incubated in a 1% solution of trimethyltetra Zolium chloride to visualize the infarcted myocardium Example 8 located within the risk region. Infarct Size is calculated by Treatment of Vascular Stroke Summing the values for each left ventricular Slice and is 65 A patient has just been diagnosed with acute multiple further expressed as a fraction of the risk region of the left vascular Strokes and is comatose. Immediately, a physician Ventricle. or a nurse parenterally administers a compound of Formula US 6,197,785 B1 67 68 I, either as a Single dose or as a Series of divided doses. Due Example 12 to the comatose State of the patient, the patient also receives In vivo Radiosensitization the same or a different PARP inhibitor by intermittent or Before undergoing radiation therapy to treat cancer, a continuous administration via implantation of a patient is administered an effective amount of a compound biocompatible, biodegradable polymeric matrix delivery or a pharmaceutical composition of the present invention. System comprising a compound of Formula I, or via a The compound or pharmaceutical composition acts as a Subdural pump inserted to administer the compound directly radioSensitizer and making the tumor more Susceptible to to the infarct area of the brain. It is expected by the inventors radiation therapy. that the patient would awaken from the coma more quickly Example 13 than if the compound of the invention were not adminis Measuring Altered Gene Expression in mRNA Senescent tered. The treatment is also expected to reduce the Severity Cells of the patient's residual neurological Symptoms. In addition, Human fibroblast BJ cells, at Population Doubling (PDL) it is expected that re-occurrence of vascular Stroke would be 94, are plated in regular growth medium and then changed reduced. to low Serum medium to reflect physiological conditions Example 9 15 described in Linskens, et al., Nucleic Acids Res. Preventing Cardiac Reperfusion Injury 23:16:3244-3251 (1995). A medium of DMEM/199 A patient is diagnosed with life-threatening cardiomyopa wupplemented with 0.5% bovine calf serum is used. The thy and requires a heart transplant. Until a donor heart is cells are treated daily for 13 days with the PARP inhibitor of found, the patient is maintained on EXtra Corporeal Oxy Formula I as disclosed herein. The control cells are treated genation Monitoring (ECMO). with and without the Solvent used to administer the PARP A donor heart is then located, and the patient undergoes a inhibitor. The untreated old and young control cells are Surgical transplant procedure, during which the patient is tested for comparison. RNA is prepared from the treated and placed on a heart-lung pump. The patient receives a com control cells according to the techniques described in PCT pound of the invention intracardiac within a specified period 25 Publication No. 96/13610 and Northern blotting is con of time prior to re-routing his or her circulation from the ducted. Probes Specific for Senescence-related genes are heart-lung pump to his or her new heart, thus preventing analyzed, and treated and control cells compared. In ana cardiac reperfusion injury as the new heart begins to beat lyzing the results, the lowest level of gene expression is independently of the external heart-lung pump. arbitrarily Set at 1 to provide a basis for comparison. Three genes particularly relevant to age-related changes in the skin Example 10 are collagen, collagenase and elastin. West, Arch. Derm. Septic Shock Assay 130:87–95 (1994). Elastin expression of the cells treated Groups of 10 C57/BL male mice weighing 18 to 20 g were with the PARP inhibitor of Formula I is significantly administered a test compound, 1-carboxynaphthalene-1- increased in comparison with the control cells. Elastin carboxamide at the doses of 60, 20, 6 and 2 mg/kg, daily, by expression is significantly higher in young cells compared to intraperitoneal (IP) injection for three consecutive days. 35 Senescent cells, and thus treatment with the PARP inhibitor Each animal was first challenged with lipopolysaccharide of Formula I causes elastin expression levels in Senescent (LPS, from E. Coli, LDoo of 20 mg/animal IV) plus cells to change to levels similar to those found in much galactosamine (20 mg/animal IV). The first dose of test younger cells. Similarly, a beneficial effect is Seen in colla compound in a Suitable vehicle was given 30 minutes after genase and collagen expression with treatment with the challenge, and the Second and third doses were given 24 40 PARP inhibitors of Formula I. hours later on day 2 and day 3 respectively, with only the Surviving animals receiving the Second or third dose of the Example 14 test compound. Mortality was recorded every 12 hours after Measuring Altered Gene Expression Protein in Senescent challenge for the three-day testing period. 1-Carboxy Cells naphthalene-1-carboxamide provided a protection against 45 Approximately 105 BJ cells, at PDL 95-100 are plated mortality from Septic shock of about 40%. Based on these and grown in 15 cm dishes. The growth medium is DMEM/ results, other compounds of the invention are expected to 199 Supplemented with 10% bovice calf serum. The cells are provide a protection against mortality exceeding about 35%. treated daily for 24 hours with the PARP inhibitors of Formula I (100 lug/1 mL of medium). The cells are washed Example 11 50 with phosphate buffered solution (PBS), then permeablized In vitro Radiosensitization with 4% paraformaldehyde for 5 minutes, then washed with The human prostate cancer cell line, PC-3s, were plated in PBS, and treated with 100% cold methanol for 10 minutes. 6 well dishes and grown at monolayer cultures in RPMI1640 The methanol is removed and the cells are washed with PBS, supplemented with 10% FCS. The cells are maintained at and then treated with 10% serum to block nonspecific 37° C. in 5% CO and 95% air. The cells were exposed to 55 antibody binding. About 1 mL of the appropriate commer a dose response (0.1 mM to 0.1 uM) of 3 different PARP cially available antibody solutions (1:500 dilution. Vector) is inhibitors of Formula I disclosed herein prior to irradiation added to the cells and the mixture incubated for 1 hour. The at one Sublethal dose level. For all treatment groups, the Six cells are rinsed and washed three times with PBS. A sec well plates were exposed at room temperature in a Seifert ondary antibody, goat anti-mouse IgG (1 mL) with a biotin 250 kV/15 mA irradiator with a 0.5 mm Cu/l mm. Cell 60 tag is added along with 1 mL of a Solution containing viability was examined by exclusion of 0.4% trypan blue. Streptavidin conjugated to alkaline phosphatase and 1 mL of Dye exclusion was assessed Visually by microScopy and NBT reagent (Vector). The cells are washed and changes in viable cell number was calculated by Subtracting the number gene expression are noted calorimetrically. Four Senescence of cells from the viable cell number and dividing by the total Specific genes-collagen I, collagen III, collagenase, and number of cells. Cell proliferation rates were calculated by 65 interferon gamma-in senescent cells treated with the PARP the amount of H-thymidine incorporation post-irradiation. inhibitor of Formula I are monitored and the results show a The PARP inhibitors show radiosensitization of the cells. decrease in interferon gamma expression with no observable US 6,197,785 B1 69 70 change in the expression levels of the other three genes, Dark neurons are observed bilaterally within the spinal demonstrating that the PARP inhibitors of Formula I can cord dorsal horn, particularly in laminae I-II, of rats 8 days alter Senescence-specific gene expression. after unilateral Sciatic nerve ligation as compared with Sham Example 15 operated rats. Various doses of differing compounds of Extending or Increasing Proliferative Capacity and Lifespan Formula I are tested in this model and show that the Formula of Cells I compounds reduce both incidence of dark neurons and To demonstrate the effectiveness of the present method neuropathic pain behavior in CCI rats. for extending the proliferative capacity and lifespan of cells, The invention being thus described, it will be obvious that human fibroblast cells lines (either W138 at Population the same may be varied in many ways. Such variations are Doubling (PDL) 23 or BJ cells at PDL 71) are thawed and not to be regarded as a departure from the Spirit and Scope plated on T75 flasks and allowed to grow in normal medium of the invention, and all Such modifications are intended to (DMEM/M199 plus 10% bovine calf serum) for about a be included within the scope of the following claims. week, at which time the cells are confluent, and the cultures We claim: are therefor ready to be subdivided. At the time of 1. A method of treating a cardiovascular disorder in an Subdivision, the media is aspirated, and the cells rinsed with 15 animal comprising administering to Said animal an effective phosphate buffer saline (PBS) and then trypsinized. The amount of a compound of formula I: cells are counted with a Coulter counter and plated at a density of 10 cells per cm in 6-well tissue culture plates in DMEM/199 medium supplemented with 10% bovine calf serum and varying amounts (0.10 uM, and 1 mM: from a 100xstock solution in DMEM/M199 medium) of a PARP inhibitor of Formula I as disclosed herein. This process is repeated every 7 days until the cell appear to Stop dividing. The untreated (control) cells reach Senescence and stop dividing after about 40 days in culture. Treatment of cells with 10 uM 3-AB appears to have little or no effect in 25 or a pharmaceutically acceptable Salt, hydrate, ester, Solvate, contrast to treatment with 100 uM 3-AB which appears prodrug, metabolite, Stereoisomer, or mixtures thereof, lengthen the lifespan of the cells and treatment with 1 mM wherein: 3-AB which dramatically increases the lifespan and prolif R", when present, is hydrogen or lower alkyl; erative capacity of the cells. The cells treated with 1 mM R is lower alkyl, aryl, aralkyl, lower alkanoyl, or 3-AB will still divide after 60 days in culture. -(CH), (CHOH),(CH), A, wherein n is 1-4, y is 0 Example 16 or 1, m is 0-5, and A is cycloalkyl, cycloalkenyl, lower Neuroprotective Effects of Formula I on Chronic Constric alkanoyl, aryl, aralkyl, -NH, -NH-(lower alkyl), tion Injury (CCI) in Rats Adult male Sprague-Dawley rats, 300-350 g, are anes 35 / lower alkwlyl) thetized with intraperitoneal 50 mg/kg Sodium pentobarbital. -N Nerve ligation is performed by exposing one side of the rat's (lower alkyl), Sciatic nerves and dissecting a 5-7 mm-long nerve Segment -O). -O) O and closing with four loose ligatures at a 1.0–1.5-mm, followed by implanting of an intrathecal catheter and insert 40 ing of a gentamicin sulfate-flushed polyethylene (PE-10) -O tube into the Subarachnoid Space through an incision at the cisterna magna. The caudal end of the catheter is gently threaded to the lumbar enlargement and the rostral end is Y represents the atoms necessary to form a fused 5- to Secured with dental cement to a Screw embedded in the skull 45 6-membered ring that is aromatic or nonaromatic and and the skin wound is closed with wound clips. carbocyclic, which may be Substituted or unsubstituted; Thermal hyperalgesia to radiant heat is assessed by using Z is (i) —CHRCHR- where R and R are indepen a paw-withdrawal test. The rat is placed in a plastic cylinder dently hydrogen, alkyl, aryl or aralkyl, or on a 3-mm thick glass plate with a radiant heat Source from (ii)—RC=CR- where R and Rare independently a projection bulb placed directly under the plantar Surface of 50 hydrogen, lower alkyl, aryl, aralkyl, chlorine, bromine the rat’s hindpaw. The paw-withdrawal latency is defined as or -NRR, where R7 and R are independently the time elapsed from the onset of radiant heat Stimulation hydrogen or lower alkyl, or, Rand R, taken together, to withdrawal of the rat’s hindpaw. form a fused 5- to 6-membered ring that is aromatic or Mechanical hyperalgesia is assessed by placing the rat in nonaromatic and carbocyclic or heterocyclic. a cage with a bottom made of perforated metal sheet with 55 2. The method of claim 1, wherein the cardiovascular many Small Square holes. Duration of paw-withdrawal is disorder is coronary artery disease, myocardial infarction, recorded after pricking the mid-plantar Surface of the rat's angina pectoris, cardiogenic Shock or cardiovascular tissue hindpaw with the tip of a Safety pin inserted through the cage damage. bottom. 3. The method of claim 1 wherein Y is a 6 membered Mechano-allodynia is assessed by placing a rat in a cage 60 aromatic carbocyclic ring. Similar to the previous test, and applying von Frey filaments 4. The method of claim 3 wherein Y contains at least one in ascending order of bending force ranging from 0.07 to 76 Substitueut. g to the mid-plantar Surface of the rats hindpaw. A Von Frey 5. The method of claim 4 wherein said at least one filament is applied perpendicular to the skin and depressed Substitueut is Selected from the group consisting of an alkyl slowly until it bends. A threshold force of response is defined 65 group, an alkenyl group, an alkynyl group, an alkanoyl as the first filament in the Series to evoke at least one clear group, a cycloalkyl group, a cycloalkenyl group, an arakyl paw-withdrawal out of five applications. group and an aryl group. US 6,197,785 B1 71 72 6. The method of claim 5 wherein R is -(CH), 18. The method of claim 3 wherein A is cycloalkyl, (CHOH)-(CH), A. cycloalkenyl, lower alkanoyl, aryl, aralkyl, 7. The method of claim 6 wherein A is aralkyl. 8. The method of claim 7 wherein said compound is 1-(benzyloxy)-5-methylisoquinoline. 9. The method of claim 1 wherein said compound is Selected from the group consisting of 1-(methoxy-)-5- methylisoquinoline, 1-(ethoxy-)-5-methylisoquinoline, 1-(propoxy)-5-methylisoquinoline, 1-(butoxy)-5- methylisoquinoline, 1-(ethoxy)-5-hydroxyisoquinoline, 19. The method of claim 4 wherein A is cycloakyl, 1-(propoxy)-5-hydroxyisoquinoline and 1-(butoxy)-5- cycloalkonyl, lower alkanoyl, aryl, aralkyl, hydroxyisoquinoline. 10. The method of claim 3, wherein the cardiovascular disorder is coronary artery disease, myocardial infarction, angina pectoris, cardiogenic Shock or cardiovascular tissue 15 damage. 11. The method of claim 4, wherein the cardiovascular disorder is coronary artery disease, myocardial infarction, 20. The method of claim 5 wherein A is cycloalkyl, angina pectoris, cardiogenic Shock or cardiovascular tissue cycloalkenyl, lower alkanoyl, aryl, aralkyl, damage. 12. The method of claim 5, wherein the cardiovascular disorder is coronary artery disease, myocardial infarction, angina pectoris, cardiogenic Shock or cardiovascular tissue damage. 13. The method of claim 6, wherein the cardiovascular 25 disorder is coronary artery disease, myocardial infarction, 21. The method of claim 6 wherein A is cycloalkyl, angina pectoris, cardiogenic Shock or cardiovascular tissue cycloalkenyl, lower alkanoyl, aryl, aralkyl, damage. 14. The method of claim 7, wherein the cardiovascular disorder is coronary artery disease, myocardial infarction, angina pectoris, cardiogenic Shock or cardiovascular tissue damage. 15. The method of claim 8, wherein the cardiovascular disorder is coronary artery disease, myocardial infarction, 22. The method of claim 18 wherein the cardiovascular angina pectoris, cardiogenic Shock or cardiovascular tissue 35 damage. disorder is coronary artery disease, myocardial infraction, 16. The method of claim 9, wherein the cardiovascular angina pectoris, cardiogenic Shock or cardiovascular tissue disorder is coronary artery disease, myocardial infarction, damage. angina pectoris, cardiogenic Shock or cardiovascular tissue 23. The method of claim 18 wherein said compound has damage. 40 an ICso of 100 um or lower for inhibiting poly (ADP-ribose) 17. The method of claim 1 wherein said compound has an polymerase in vitro. ICs of 100 um or lower for inhibiting poly (ADP-ribose) polymerase in vitro.